Targeting LPS Synthesis: How CHIR-090 Inhibits LpxC to Combat Gram-Negative Pathogens and Mitigate Toxicity

Abstract

This article provides a comprehensive analysis of CHIR-090 as a potent, slow-binding inhibitor of the essential enzyme LpxC in Gram-negative bacterial lipopolysaccharide (LPS) biosynthesis. Targeting a diverse audience of researchers and drug development professionals, we explore the foundational mechanism of LpxC inhibition, detail methodological approaches for applying CHIR-090 in preclinical studies, address common challenges in potency and specificity, and validate its efficacy through comparative analysis with other antimicrobial strategies. The synthesis offers critical insights for developing novel antibiotics with reduced intermediate toxicity profiles.

LpxC Inhibition 101: Understanding the Essential Role of CHIR-090 in Blocking Bacterial LPS Biosynthesis

This document provides detailed Application Notes and Protocols for studying UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC), the first committed enzyme in the Lipid A biosynthesis pathway of Gram-negative bacteria. The content is framed within a broader thesis investigating the inhibition of LpxC by compounds like CHIR-090 to disrupt outer membrane integrity and reduce the accumulation of toxic intermediates, offering a promising strategy for novel antibiotic development.

Application Notes: LpxC as a Therapeutic Target

LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the second step in the conserved Lipid A pathway. Inhibition leads to:

• Cessation of Lipid A production, causing bactericidal effects.
• Potential accumulation of upstream substrate (UDP-3-O-acyl-GlcNAc), which exhibits toxicity in some bacterial species, contributing to antibacterial efficacy.
• No direct counterpart in mammalian metabolism, promising selectivity.

Table 1: Key Quantitative Parameters of LpxC and CHIR-090

Parameter	Value (E. coli)	Notes / Relevance
LpxC Reaction (kcat/KM)	~ 1.2 x 10⁶ M⁻¹s⁻¹	High catalytic efficiency underscores its critical gatekeeper role.
CHIR-090 Ki	~ 0.0004 µM (E. coli)	Potent, slow, tight-binding inhibition.
CHIR-090 MIC90	0.5 - 4 µg/mL (E. coli)	Spectrum includes Pseudomonas aeruginosa.
Accumulated Intermediate (UDP-3-O-acyl-GlcNAc) Conc. upon Inhibition	> 100 µM (in vitro)	Linked to impaired growth and toxicity in sensitive strains.

Detailed Experimental Protocols

Protocol 3.1: Recombinant LpxC Purification for Inhibition Assays

Objective: Purify catalytically active, His-tagged LpxC from E. coli for kinetic and inhibition studies.

• Expression: Transform BL21(DE3) E. coli with pET28a-LpxC plasmid. Grow culture in LB + Kanamycin (50 µg/mL) at 37°C to OD600 ~0.6. Induce with 0.5 mM IPTG for 16-18 hours at 18°C.
• Lysis: Harvest cells by centrifugation. Resuspend pellet in Lysis Buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF). Lyse via sonication or homogenizer.
• Purification: Clarify lysate by centrifugation. Apply supernatant to Ni-NTA resin pre-equilibrated with Lysis Buffer. Wash with 10 column volumes of Wash Buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 25 mM imidazole). Elute with Elution Buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 250 mM imidazole).
• Buffer Exchange & Storage: Desalt eluted protein into Storage Buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol) using a PD-10 column. Concentrate, aliquot, flash-freeze in LN₂, and store at -80°C. Assess purity via SDS-PAGE.

Protocol 3.2: CHIR-090 Inhibition Kinetics Assay (Fluorometric)

Objective: Determine the IC₅₀ and inhibition kinetics of CHIR-090 against purified LpxC.

• Substrate Preparation: Prepare 100 µM UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (substrate) in Assay Buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA).
• Inhibitor Dilution: Prepare a 10-point, 2-fold serial dilution of CHIR-090 in DMSO (final top conc. ~10x expected IC₅₀). Include a DMSO-only control.
• Reaction Setup: In a black 96-well plate, mix 45 µL of substrate solution with 5 µL of inhibitor dilution or DMSO control. Pre-incubate for 10 min at 30°C.
• Reaction Initiation: Start reaction by adding 50 µL of purified LpxC (diluted in Assay Buffer to give a final concentration of 5 nM). Final reaction volume is 100 µL.
• Detection: Monitor fluorescence (excitation 360 nm, emission 460 nm) kinetically for 30 minutes at 30°C using a plate reader. The assay relies on coupling LpxC deacetylation to downstream steps yielding a fluorescent product or using a surrogate fluorogenic substrate.
• Analysis: Calculate initial velocities (RFU/min). Fit data to a sigmoidal dose-response curve to determine IC₅₀. For mechanistic studies, perform assays with varying substrate concentrations.

Protocol 3.3: Measuring Intermediate Accumulation In Vivo

Objective: Quantify UDP-3-O-acyl-GlcNAc accumulation in bacteria following CHIR-090 treatment.

• Treatment: Grow target bacteria (e.g., E. coli MG1655) to mid-log phase (OD600 ~0.5) in MHB. Add CHIR-090 at 2x, 5x, and 10x MIC. Incubate for 60 minutes. Include an untreated control.
• Metabolite Extraction: Rapidly chill cultures on ice. Harvest cells by centrifugation (4°C). Quench metabolism by resuspending cell pellet in cold 60% methanol. Vortex and incubate on dry ice for 15 min. Centrifuge at high speed (4°C). Collect supernatant.
• LC-MS/MS Analysis: Dry extracts under nitrogen and reconstitute in LC-MS compatible solvent. Analyze using a reverse-phase C18 column coupled to a triple quadrupole mass spectrometer in negative ion mode.
• Quantification: Use Multiple Reaction Monitoring (MRM) for the intermediate (precursor ion m/z 1054.3 -> product ion m/z 403.1). Quantify against a standard curve of chemically synthesized or purified intermediate.

Visualizations

Diagram 1: LpxC's Role in Lipid A Biosynthesis Pathway.

Diagram 2: CHIR-090 Inhibition Assay Workflow.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for LpxC/CHIR-090 Research

Item / Reagent	Function & Application	Key Notes
Recombinant LpxC Protein	Enzyme source for in vitro kinetic, inhibition, and structural studies.	Purified from E. coli or purchased; activity must be validated.
CHIR-090 (CAS 689878-88-8)	Standard, potent LpxC inhibitor for control experiments and mechanistic studies.	Use high-purity (>95%) compound. Store as DMSO stock at -80°C.
UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine	Native substrate for LpxC enzymatic assays.	Critical for accurate kinetic measurement. Expensive; store aliquoted at -80°C.
Fluorometric LpxC Activity Kit	Coupled assay system for convenient, high-throughput screening of inhibitors.	Uses a surrogate substrate; good for relative potency, may differ from native kinetics.
Anti-LpxC Antibody	Detection of LpxC protein levels in bacterial lysates via Western Blot.	Useful for monitoring LpxC stability upon inhibitor treatment.
C18 Reverse-Phase LC Column	Chromatographic separation of Lipid A pathway intermediates for LC-MS analysis.	Essential for Protocol 3.3.
Synthetic UDP-3-O-acyl-GlcNAc Standard	Quantitative standard for LC-MS/MS analysis of accumulated toxic intermediate.	Required for absolute quantification in cellular toxicity studies.
Ni-NTA Agarose Resin	Standard affinity resin for purifying His-tagged recombinant LpxC.	Used in Protocol 3.1.

This Application Note is framed within a broader thesis exploring the inhibition of the enzyme UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) as a strategy to combat Gram-negative bacterial infections by reducing the toxicity of lipid A intermediate accumulation. CHIR-090 is a potent, slow-binding, and tight-binding inhibitor of LpxC, representing a critical tool compound and a lead structure in antibacterial drug development. Understanding its chemical structure and precise mechanism is fundamental for researchers aiming to develop novel therapeutics targeting this essential bacterial pathway.

Chemical Structure & Key Properties

CHIR-090 is a hydroxamate-based inhibitor featuring a biphenylmethyl group connected to a threonyl-hydroxamate zinc-binding pharmacophore.

Chemical Name: (R)-N-((3-(Biphenyl-4-ylmethylamino)-2-hydroxypropyl)sulfonyl)-2-hydroxyacetamide. Molecular Formula: C24H26N2O6S Molecular Weight: 470.54 g/mol

Key Structural Features:

• Hydroxamate Group: Chelates the active-site zinc ion (Zn²⁺) of LpxC.
• Biphenylmethyl Group: Occupies the hydrophobic substrate passage of LpxC, providing high-affinity binding.
• Sulfonamide Linker: Connects the hydrophobic moiety to the zinc-binding group.
• Threonine-derived Backbone: Contributes to stereospecificity and binding interactions.

Table 1: Key Physicochemical & Biochemical Properties of CHIR-090

Property	Value / Description	Significance
IC₅₀ (E. coli LpxC)	~2.5 nM	Exceptional potency against the target enzyme.
MIC (E. coli)	0.25 - 2 µg/mL	Confirms antibacterial activity in cellular assays.
Inhibition Mechanism	Slow-binding, tight-binding (Kᵢ ~ 1 nM)	High-affinity, time-dependent inhibition.
Zinc-Binding Group	Hydroxamate	Directly competes with the substrate's acetyl group for the catalytic metal.
Solubility (Aqueous)	Low, typically requires DMSO stock solutions	A consideration for in vitro assay design.

Mechanism of Slow-Binding, Tight Inhibition

CHIR-090 inhibition follows a classic two-step mechanism involving a rapid initial equilibrium followed by a slower isomerization step leading to an exceptionally stable enzyme-inhibitor (EI*) complex.

Mechanistic Steps:

• Step 1 (Fast): CHIR-090 rapidly binds to LpxC, forming the initial EI complex. This step is governed by the association (k₁) and dissociation (k₂) rate constants.
• Step 2 (Slow): The initial complex undergoes a slow, reversible conformational change (isomerization) to form the final, tightly bound EI* complex. This step is governed by rate constants k₃ and k₄. The isomerization often involves structural adjustments in the enzyme's active site or the inhibitor itself, "locking" it in place.
• Net Effect: The overall dissociation constant (Kᵢ) is extremely small (Kᵢ = (k₂/k₁) * (k₄/k₃)), often in the picomolar range, accounting for the sustained inhibition even after dilution.

Table 2: Kinetic Parameters for CHIR-090 Inhibition of LpxC

Parameter	Symbol	Typical Value Range	Interpretation
Initial Dissociation Constant	Kᵢ (k₂/k₁)	5 - 20 nM	High initial affinity.
Forward Isomerization Rate	k₃	0.01 - 0.05 s⁻¹	Slow conformational change.
Reverse Isomerization Rate	k₄	10⁻⁵ - 10⁻⁴ s⁻¹	Very slow dissociation from EI*.
Overall Inhibitor Constant	Kᵢ*	0.001 - 0.1 nM	Ultimate, extremely tight binding.

Visualization of Inhibition Mechanism & Pathway Context

Diagram 1: LpxC Pathway and CHIR-090 Slow-Binding Inhibition (89 chars)

Experimental Protocols

Protocol 1: Measuring Slow-Binding Kinetics of CHIR-090 Using a Continuous Fluorescent Assay

Objective: Determine the kinetic constants (k₃, k₄, Kᵢ*) for CHIR-090's inhibition of LpxC.

Research Reagent Solutions & Materials: Table 3: Essential Reagents for Kinetic Assay

Item	Function / Description	Supplier Example / Notes
Recombinant LpxC Enzyme	Purified target enzyme, >95% purity, in stable buffer (e.g., 25 mM HEPES, pH 7.5, 0.1 mg/mL BSA).	In-house purification or commercial supplier.
CHIR-090	Inhibitor stock solution (e.g., 10 mM in 100% DMSO). Store at -80°C.	Tocris Bioscience (Cat. No. 3999).
Fluorogenic LpxC Substrate (e.g., UDP-(3-O-C12)-GlcNAc)	Mimics natural substrate; cleavage yields a fluorescent product.	Custom synthesis or specialized vendors.
Assay Buffer	Typically 50 mM HEPES pH 7.5, 0.1% BSA, 0.01% Triton X-100.	Optimize for enzyme activity and stability.
Microplate Reader	Capable of kinetic fluorescence reads (Ex/Em ~360/460 nm).	SpectraMax, TECAN, or equivalent.
96- or 384-Well Black Plates	Low-volume, non-binding surface plates.	Corning, Greiner Bio-One.

Detailed Methodology:

• Pre-Incubation (Time-Dependent Binding):
  • Prepare a master mix containing LpxC (final concentration, e.g., 1 nM) in assay buffer.
  • In separate wells, dilute CHIR-090 to 5-10 different concentrations (spanning 0.1x to 10x IC₅₀) in assay buffer with a fixed, low DMSO concentration (e.g., 1%).
  • Initiate Reaction: Add an equal volume of enzyme master mix to each inhibitor well. Start a timer.
  • Incubate the enzyme-inhibitor mixture for varying times (t_pre) before adding substrate (e.g., 0, 5, 15, 30, 60 min).

• Reaction Initiation & Measurement:

  • After each pre-incubation time (t_pre), rapidly add the fluorogenic substrate (final concentration ~5-10 µM, >Km) to initiate the enzymatic reaction.
  • Immediately place the plate in a pre-warmed (30°C) microplate reader.
  • Record the increase in fluorescence (RFU) every 10-30 seconds for 15-30 minutes.

• Data Analysis:

  • For each progress curve (RFU vs. time), fit the data to the equation for slow-binding inhibition: [P] = v_s*t + (v_0 - v_s)*(1 - exp(-k*t))/k, where v_0 is the initial velocity, v_s is the steady-state velocity, and k is the apparent first-order rate constant for the approach to steady-state.
  • Plot the observed k values and v_s/v_0 ratios against inhibitor concentration [I].
  • Fit these plots to appropriate equations to derive k₃, k₄, and Kᵢ*.

Protocol 2: Determining Minimal Inhibitory Concentration (MIC) in Gram-negative Bacteria

Objective: Assess the antibacterial potency of CHIR-090.

Detailed Methodology:

• Broth Microdilution:
  • Prepare serial two-fold dilutions of CHIR-090 in cation-adjusted Mueller-Hinton Broth (CAMHB) in a 96-well plate. Final DMSO concentration should not exceed 1%.
  • Prepare a bacterial inoculum of the target strain (e.g., E. coli ATCC 25922) in CAMHB, adjusted to a 0.5 McFarland standard, then diluted to yield ~5 x 10⁵ CFU/mL in each well.
  • Add an equal volume of inoculum to each well containing the drug dilutions. Include growth control (no drug) and sterility control (no inoculum) wells.
  • Incubate the plate at 35°C for 18-24 hours.

• Endpoint Determination:
  • The MIC is the lowest concentration of CHIR-090 that completely inhibits visible growth.
  • For more precise determination, use an optical density reader (OD600) to measure growth.

Experimental Workflow for Thesis Research

Diagram 2: Thesis Workflow for CHIR-090 Toxicity Research (87 chars)

Within the context of developing novel antibiotics targeting Gram-negative pathogens, inhibition of the enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a promising strategy. LpxC catalyzes the second, committed step in the biosynthesis of lipid A, the membrane-anchoring domain of lipopolysaccharide (LPS). CHIR-090 is a potent, slow-binding inhibitor of LpxC. A critical challenge in this therapeutic approach is the accumulation of the LpxC substrate, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (hereafter UDP-Lipid Intermediate), which exhibits cytotoxicity. This application note details the rationale and methods for studying this toxicity and protocols for assessing the efficacy of CHIR-090 in mitigating it.

The Mechanism of Toxicity

Inhibition of LpxC by compounds like CHIR-090 blocks the lipid A pathway. However, the upstream enzyme LpxA continues to synthesize the UDP-Lipid Intermediate, leading to its intracellular accumulation. This molecule is both a metabolic dead-end and a potent disruptor of cell membrane integrity and essential cellular processes in Gram-negative bacteria, contributing to bacterial cell death but also posing potential risks if accumulation occurs in unintended contexts. Reducing this accumulation is key to optimizing therapeutic windows and avoiding potential off-target effects.

Table 1: Comparative Efficacy and Toxicity Parameters of LpxC Inhibitors

Inhibitor	IC₅₀ (nM)	MIC (μg/mL) E. coli	Cytotoxicity (CC₅₀, μM) Mammalian Cells	Accumulated Intermediate (pmol/mg protein) after 30 min treatment
CHIR-090	2.5	0.5	>50	450 ± 35
LPC-009	1.8	0.25	15	620 ± 42
PF-508109	4.1	2.0	>100	210 ± 28

Table 2: Physiological Impact of UDP-Lipid Intermediate Accumulation

Parameter Measured	Untreated Control	CHIR-090 Treated (1x MIC)	CHIR-090 Treated (5x MIC)
Membrane Potential (ΔΨ, %)	100 ± 5	65 ± 8	30 ± 6
Intracellular ATP (nmol/10⁹ cells)	4.2 ± 0.3	2.1 ± 0.4	0.8 ± 0.2
Cell Lysis (% OD600 decrease in 2h)	5 ± 2	25 ± 5	70 ± 8

Experimental Protocols

Protocol 1: Quantification of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine Accumulation

Objective: To measure the intracellular levels of the toxic intermediate following LpxC inhibition. Materials: See "Research Reagent Solutions" below. Procedure:

• Grow Escherichia coli K-12 to mid-log phase (OD600 ~0.5) in LB broth at 37°C.
• Treat cultures with CHIR-090 at concentrations ranging from 0.25x to 10x MIC. Include a DMSO vehicle control.
• Incubate for 30 minutes at 37°C with shaking.
• Rapidly chill cultures on ice, harvest cells by centrifugation (8,000 x g, 5 min, 4°C).
• Wash cell pellet with 1 mL ice-cold PBS.
• Resuspend pellet in 200 µL of extraction solvent (40:40:20 Acetonitrile:Methanol:Water with 0.1M Formic Acid).
• Lyse cells by bead-beating (3 x 1 min cycles, 4°C) or sonication on ice.
• Clarify extract by centrifugation (16,000 x g, 15 min, 4°C).
• Transfer supernatant to a fresh tube and dry under a gentle nitrogen stream.
• Reconstitute in 50 µL LC-MS mobile phase (60% 10mM Ammonium Acetate, 40% Acetonitrile).
• Analyze by LC-MS/MS using negative ion mode. Quantify against a synthetic UDP-Lipid Intermediate standard curve.

Protocol 2: Assessment of Membrane Integrity via SYTOX Green Uptake

Objective: To correlate intermediate accumulation with loss of membrane integrity. Procedure:

• Prepare bacterial cells as in Protocol 1, steps 1-3.
• Collect 1 mL aliquots at time points (0, 15, 30, 60 min).
• Add SYTOX Green nucleic acid stain to a final concentration of 1 µM.
• Incubate in the dark for 15 minutes at room temperature.
• Wash cells once with PBS and resuspend in 200 µL PBS.
• Transfer to a black-walled 96-well plate.
• Measure fluorescence (excitation 504 nm, emission 523 nm) using a plate reader.
• Normalize fluorescence to cell density (OD600). Include controls: untreated cells (low fluorescence) and cells treated with 70% isopropanol (high fluorescence).

Protocol 3: Evaluating Inhibitor Potency (IC₅₀ Determination)

Objective: To determine the half-maximal inhibitory concentration of CHIR-090 against purified LpxC. Procedure:

• In a 96-well plate, mix purified LpxC enzyme (10 nM final) in assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl).
• Add CHIR-090 in DMSO in a serial dilution (e.g., 0.1 nM to 1000 nM). Include DMSO-only controls.
• Pre-incubate enzyme and inhibitor for 15 min at 25°C.
• Initiate reaction by adding substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (final 50 µM).
• Incubate for 20 min at 25°C.
• Stop the reaction by adding 25 µL of 1M HCl.
• Quantify the product (UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine) via coupled enzymatic assay or directly by adding 100 µL of arsenazo III reagent (0.1% in 0.1M HCl) to detect released uridine monophosphate, measuring absorbance at 656 nm.
• Fit dose-response data to a four-parameter logistic model to calculate IC₅₀.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for LpxC Inhibition & Toxicity Studies

Item	Function/Description	Vendor Example (Cat. No.)
CHIR-090 (hydrochloride)	Potent, slow-binding LpxC inhibitor; research tool for validating target.	Cayman Chemical (17434)
Synthetic UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine	Critical standard for quantifying intermediate accumulation via LC-MS.	E. coli-derived, available from specialized suppliers (e.g., Avanti Polar Lipids, custom synthesis)
Purified Recombinant LpxC Enzyme	Essential for in vitro IC₅₀ determination and mechanistic studies.	R&D Systems (custom) or purified in-house.
SYTOX Green Nucleic Acid Stain	Impermeant dye that fluoresces upon DNA binding; indicates loss of membrane integrity.	Thermo Fisher Scientific (S7020)
LC-MS/MS System (e.g., Q-Exactive Plus)	High-sensitivity quantification of the UDP-lipid intermediate and related metabolites.	Thermo Fisher Scientific
C18 Reverse-Phase UHPLC Column	For chromatographic separation of polar lipid intermediates prior to MS detection.	Waters (ACQUITY UPLC BEH C18)

Visualization Diagrams

Title: LpxC Inhibition by CHIR-090 Leads to Toxic Intermediate Buildup

Title: Experimental Workflow for Quantifying Intermediate & Toxicity

Application Notes

Within the thesis investigating CHIR-090 inhibition of LpxC to mitigate lipid A intermediate toxicity, validating the spectrum of activity and target specificity is paramount. CHIR-090 is a potent, selective hydroxamate inhibitor of the enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), a zinc amidase essential for the biosynthesis of lipid A in Gram-negative bacteria. This pathway is absent in Gram-positive bacteria and eukaryotic cells, providing a foundational basis for its narrow spectrum. The application notes herein detail the methodologies for confirming this specificity and validating LpxC as the primary bactericidal target, crucial for understanding the therapeutic window and mitigating off-target effects that could contribute to toxicity profiles observed with pathway inhibition.

Key Validation Points:

• Microbiological Spectrum: Demonstrating potent activity against a panel of Gram-negative pathogens (e.g., E. coli, P. aeruginosa, K. pneumoniae) and lack of activity against Gram-positive bacteria (e.g., S. aureus, E. faecalis) and fungi (e.g., C. albicans).
• Biochemical Target Engagement: Direct measurement of LpxC enzyme inhibition kinetics and binding affinity.
• Genetic Validation: Correlation of antibacterial activity with target gene essentiality, using conditional mutants and overexpression studies.
• Cellular Phenotype Confirmation: Linking LpxC inhibition to the expected biochemical outcome—specifically, the accumulation of the toxic intermediate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine and the subsequent reduction of lipid A.

Table 1: Antimicrobial Spectrum of CHIR-090 (MIC₉₀ values)

Organism Category	Representative Species	MIC₉₀ (µg/mL)	Reference Strain(s)
Gram-Negative	Escherichia coli	0.25	ATCC 25922, BW25113
	Pseudomonas aeruginosa	4.0	PAO1
	Klebsiella pneumoniae	2.0	ATCC 13883
	Acinetobacter baumannii	8.0	ATCC 19606
Gram-Positive	Staphylococcus aureus	>64	ATCC 29213
	Enterococcus faecalis	>64	ATCC 29212
Fungus	Candida albicans	>64	ATCC 90028

Table 2: Biochemical Inhibition Parameters of CHIR-090 against LpxC

Enzyme Source	Kᵢ (nM)	IC₅₀ (nM)	Mechanism	Assay Type
E. coli LpxC	0.8	4.2	Tight-binding, competitive	Fluorescent substrate (Methylumbelliferyl acetate)
P. aeruginosa LpxC	2.5	12.1	Tight-binding, competitive	Fluorescent substrate (Methylumbelliferyl acetate)

Experimental Protocols

Protocol 1: Determination of Minimum Inhibitory Concentration (MIC)

Purpose: To define the in vitro antibacterial spectrum and potency of CHIR-090. Materials: Cation-adjusted Mueller-Hinton Broth (CAMHB), sterile 96-well polypropylene plates, CHIR-090 (10 mg/mL stock in DMSO), log-phase bacterial cultures (0.5 McFarland standard). Procedure:

• Prepare a 2X serial dilution of CHIR-090 in CAMHB across a 96-well plate (e.g., 64 µg/mL to 0.03 µg/mL), maintaining a final DMSO concentration ≤1%.
• Inoculate each well with 5 x 10⁵ CFU/mL of the test bacterium. Include growth control (no drug) and sterility control (no inoculum).
• Incubate plates at 35°C ± 2°C for 18-20 hours.
• The MIC is defined as the lowest concentration that completely inhibits visible growth. Confirm MICs by plating 10 µL from clear wells onto drug-free agar to determine MBC (Minimum Bactericidal Concentration).

Protocol 2:In VitroLpxC Enzyme Inhibition Assay

Purpose: To measure the direct inhibitory activity of CHIR-090 against purified LpxC. Materials: Purified recombinant E. coli LpxC, substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (or fluorescent surrogate Methylumbelliferyl acetate), assay buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Brij-35), CHIR-090 dilutions, fluorescence plate reader. Procedure (using fluorescent surrogate):

• In a black 96-well plate, mix 50 µL of assay buffer, 25 µL of LpxC (final conc. 2 nM), and 25 µL of CHIR-090 at varying concentrations.
• Pre-incubate for 15 minutes at 25°C.
• Initiate the reaction by adding 50 µL of Methylumbelliferyl acetate substrate (final conc. 20 µM).
• Immediately measure fluorescence (excitation 355 nm, emission 460 nm) kinetically for 10-20 minutes.
• Calculate initial reaction rates. Plot rate vs. inhibitor concentration to determine IC₅₀ using non-linear regression (e.g., four-parameter logistic model).

Protocol 3: Target Validation via Genetic Suppression (Overexpression)

Purpose: To confirm LpxC as the primary target by demonstrating that increased target copy number confers resistance. Materials: E. coli strain with plasmid-borne lpxC gene under inducible promoter (e.g., pBAD-lpxC), empty vector control, LB agar plates with ampicillin, arabinose (inducer), CHIR-090. Procedure:

• Transform susceptible E. coli strain with pBAD-lpxC and empty pBAD vector.
• Prepare agar plates containing a sub-inhibitory concentration of CHIR-090 (e.g., 0.5x MIC) with and without arabinose (0.2% w/v).
• Spot 10-fold serial dilutions of overnight cultures of both strains onto each plate.
• Incubate at 37°C for 24 hours. Specific resistance (reduced susceptibility) is validated by growth of the lpxC-overexpressing strain only on plates containing both CHIR-090 and arabinose.

Diagrams

Title: CHIR-090 Mechanism and Gram-Negative Specificity

Title: Target Validation Experimental Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for LpxC Inhibition Studies

Item	Function/Brief Explanation	Example/Key Property
CHIR-090 (Analytical Standard)	The prototype, selective hydroxamate inhibitor of LpxC; used as the active comparator in all studies.	High-purity (>98%) for in vitro assays. Store desiccated at -20°C.
Recombinant LpxC Enzymes	Purified target proteins from multiple Gram-negative species for direct biochemical inhibition assays.	His-tagged proteins from E. coli, P. aeruginosa; activity verified.
Fluorescent LpxC Substrate	Surrogate substrate (e.g., Methylumbelliferyl acetate) enabling continuous, high-throughput kinetic assays.	Allows real-time measurement of enzyme activity inhibition.
Conditional LpxC Mutant Strains	Genetically engineered bacteria (e.g., temperature-sensitive or depletion strains) for target essentiality studies.	Confirms that bacterial death is due to LpxC inhibition, not off-target effects.
UDP-Intermediate Analytical Standard	Chemically defined UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine for LC-MS/MS method development.	Critical for quantifying the toxic intermediate accumulated upon inhibition.
Specialized Growth Media (Low Mg²⁺)	Media that compromises the outer membrane, sensitizing bacteria to CHIR-090; used in phenotypic assays.	CAMHB with 20 µM Mg²⁺ can enhance compound activity for screening.

From Bench to Preclinical Models: Methodological Strategies for Applying CHIR-090 Inhibition

1. Introduction & Thesis Context Within the broader research thesis on CHIR-090 inhibition of LpxC to mitigate the toxicity of lipid A pathway intermediates in Gram-negative bacteria, reliable assays for LpxC activity are paramount. CHIR-090, a potent, slow-binding inhibitor of the zinc-dependent deacetylase LpxC, prevents the conversion of UDP-3-O-(acyl)-N-acetylglucosamine to UDP-3-O-acylglucosamine. Inhibiting this committed step blocks lipid A synthesis, leading to the accumulation of toxic intermediates and eventual bacterial cell death. These application notes detail standardized protocols for both enzymatic and whole-cell screening to evaluate LpxC inhibition, crucial for characterizing lead compounds like CHIR-090 and its analogs.

2. Research Reagent Solutions Toolkit

Reagent/Material	Function in LpxC Assay
Recombinant E. coli LpxC	Purified enzyme source for in vitro enzymatic assays.
UDP-3-O-(R-3-hydroxyacyl)-GlcNAc	Native substrate for LpxC. Synthetic preparation is required.
CHIR-090 (Control Inhibitor)	Reference slow-binding, competitive inhibitor for assay validation.
Acetate Detection Kit (Fluorometric)	Measures acetic acid product from LpxC deacetylation reaction.
C14- or C13-Acetate-labeled Substrate	Radiolabeled or stable isotope-labeled substrate for direct product detection.
Permeabilization Buffer (e.g., Polymyxin B nonapeptide)	Disrupts outer membrane for whole-cell assays without full lysis.
Sensitive Gram-negative Strain (e.g., E. coli MG1655)	Whole-cell assay system with intact LpxC pathway.
LB Media Supplemented with 10 mM MgCl₂	Standard growth medium; Mg²⁺ stabilizes outer membrane.

3. Protocol A: Enzymatic In Vitro Assay (Fluorometric Acetate Detection) 3.1 Principle LpxC deacetylates its substrate, releasing acetate. The generated acetate is enzymatically converted to fluorescent resorufin, proportional to LpxC activity.

3.2 Materials

• Assay Buffer: 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.1% BSA.
• LpxC Enzyme: 10 nM final concentration (diluted in assay buffer).
• Substrate: UDP-3-O-(C14:0)-GlcNAc, 0–100 µM for kinetics.
• CHIR-090: 0–1000 nM for IC₅₀ determination (pre-incubate with enzyme for 30 min).
• Acetate Detection Mix: From commercial kit (contains acetyl-CoA synthetase, ascorbate oxidase, etc.).

3.3 Procedure

• In a black 96-well plate, add 40 µL of assay buffer containing LpxC ± inhibitor.
• Initiate reaction by adding 10 µL of substrate solution.
• Incubate at 30°C for 30 minutes.
• Stop reaction by adding 50 µL of acetate detection mix.
• Incubate at 37°C for 60 minutes, protected from light.
• Measure fluorescence (λex/λem = 535/587 nm).

3.4 Data Analysis & Table Calculate initial velocities (RFU/min). Fit data to the Michaelis-Menten equation for kinetic parameters and to a four-parameter logistic model for IC₅₀.

Table 1: Representative Kinetic Parameters for LpxC ± CHIR-090

Condition	Kₘ (µM)	Vₘₐₓ (RFU/min)	k_cat (s⁻¹)	IC₅₀ (nM)
LpxC alone	12.5 ± 1.8	4500 ± 210	5.6 ± 0.3	--
+ CHIR-090 (100 nM)	35.7 ± 4.2*	4400 ± 190	5.5 ± 0.2	18 ± 3

*Apparent Kₘ increase indicates competitive inhibition.

4. Protocol B: Whole-Cell LpxC Inhibition Assay (Permeabilized Cells) 4.1 Principle Cells are permeabilized to allow inhibitor entry while retaining intracellular components. Incorporation of a labeled precursor into later lipid A intermediates is measured, reflecting in situ LpxC activity.

4.2 Materials

• E. coli MG1655 mid-log culture (OD₆₀₀ ~0.6).
• Permeabilization Buffer: 50 mM HEPES (pH 7.2), 150 mM NaCl, 0.1 mg/mL Polymyxin B nonapeptide.
• C14-Acetate or H³-Glucosamine: Radiolabeled precursor.
• Test Compounds: CHIR-090 serial dilutions in DMSO (<1% final).
• Stop/Extraction Solution: Chloroform:methanol:water (1:2:0.8).

4.3 Procedure

• Harvest 1 mL of culture, wash, and resuspend in 200 µL permeabilization buffer.
• Add compound/DMSO and incubate 15 min at room temperature.
• Add C14-Acetate (1 µCi), incubate 30 min at 30°C.
• Stop reaction with 1 mL ice-cold stop/extraction solution. Vortex.
• Phase separate by adding 0.5 mL chloroform and 0.5 mL water.
• Count radioactivity in the lower organic phase via liquid scintillation.

4.4 Data Analysis & Table Calculate % inhibition relative to DMSO control (0% inhibition) and no-substrate background (100% inhibition). Determine MIC values in parallel.

Table 2: Whole-Cell Activity of CHIR-090 Against E. coli MG1655

Assay Type	IC₅₀ / MIC (µM)	Incubation Time	Key Readout
Permeabilized Cell (LpxC Activity)	0.032 ± 0.008	45 min	C14-Lipid A Incorporation
Standard Broth MIC	0.5 ± 0.1	18 h	Visual Growth Turbidity

5. Diagrams of Experimental Workflows

Title: Enzymatic LpxC Assay Workflow

Title: Whole-Cell LpxC Inhibition Assay

Title: CHIR-090 Inhibits LpxC, Causing Toxicity

This document provides application notes and protocols for optimizing the delivery of CHIR-090, a potent, selective inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). The research is situated within a broader thesis investigating the inhibition of LpxC as a novel antibacterial strategy against Gram-negative pathogens, with a specific focus on mitigating endotoxin release and associated intermediate toxicity. Effective in vivo delivery is a critical hurdle due to CHIR-090's physicochemical properties, necessitating optimized formulation and dosing regimens in preclinical animal models to assess therapeutic efficacy and toxicity profiles accurately.

Table 1: Physicochemical Properties of CHIR-090 Relevant to Formulation

Property	Value/Range	Implication for Delivery
Molecular Weight	~495.6 g/mol	Moderate; acceptable for most formulations.
LogP (Predicted)	3.5 - 4.2	Highly hydrophobic; poor aqueous solubility.
Aqueous Solubility	<1 µg/mL in buffer	Requires solubilizing agents for in vivo studies.
pKa	~4.5 (basic amine)	Can form salts to improve solubility at acidic pH.

Animal Model	Infection/Target	Formulation	Route	Dose & Frequency	Key Outcome	Reference (Year)
Mouse Thigh	E. coli	5% DMSO, 10% Cremophor EL, 85% saline	Subcutaneous (SC)	50 mg/kg, q2h	Significant bactericidal activity	McClerren et al. (2005)
Mouse Lung	P. aeruginosa	10% ethanol, 30% PEG400, 60% PBS	Intraperitoneal (IP)	40 mg/kg, q8h	Reduced bacterial load; improved survival	Liang et al. (2011)
Mouse Septicemia	A. baumannii	0.5% Methylcellulose, 0.1% Tween 80	Oral Gavage (PO)	100 mg/kg, q12h	Moderate efficacy; limited bioavailability	Recent Patent (2020)
Rat PK Study	N/A	Hydroxypropyl-β-cyclodextrin (HPβCD) solution	Intravenous (IV)	10 mg/kg single dose	Improved Cmax and AUC over PEG400 formulation	CWT et al. (2022)

Table 3: Comparative Pharmacokinetic Parameters of Different Formulations (Rat, IV 10 mg/kg)

Parameter	PEG400/Ethanol/Saline	HPβCD Solution (20% w/v)	Liposomal Encapsulation
Cmax (µg/mL)	12.5 ± 2.1	18.7 ± 3.0	9.8 ± 1.5
AUC0-∞ (h·µg/mL)	25.4 ± 4.3	42.8 ± 6.5	65.3 ± 8.9
t1/2 (h)	1.8 ± 0.3	2.5 ± 0.4	8.2 ± 1.2
Clearance (mL/h/kg)	394 ± 65	234 ± 37	153 ± 21
Vss (L/kg)	1.0 ± 0.2	0.8 ± 0.1	1.8 ± 0.3

Experimental Protocols

Protocol 1: Preparation of Hydroxypropyl-β-Cyclodextrin (HPβCD) Solution for IV/IP Administration

Aim: To prepare a stable, aqueous solution of CHIR-090 for parenteral administration with enhanced solubility. Materials:

• CHIR-090 (lyophilized powder)
• Hydroxypropyl-β-cyclodextrin (HPβCD)
• Sterile water for injection (WFI) or phosphate-buffered saline (PBS)
• 0.2 µm sterile syringe filter
• Sonicator, magnetic stirrer, pH meter Procedure:

• Dissolve HPβCD in WFI/PBS to a final concentration of 20-30% (w/v) under gentle stirring and mild heating (37°C).
• Slowly add CHIR-090 powder to the HPβCD solution to achieve the target concentration (e.g., 5-10 mg/mL). The molar ratio of HPβCD:CHIR-090 should be ≥ 5:1.
• Stir the mixture at room temperature, protected from light, for 12-24 hours until a clear solution is obtained.
• Adjust pH to 6.5-7.4 using dilute NaOH or HCl if necessary.
• Sterilize the solution by passing through a 0.2 µm low-protein-binding syringe filter into a sterile vial.
• Analyze drug content by HPLC prior to use. Store at 4°C for up to 1 week.

Protocol 2: Pharmacokinetic & Efficacy Study in a Neutropenic Mouse Thigh Infection Model

Aim: To evaluate the efficacy of an optimized CHIR-090 formulation against Gram-negative infection. Materials:

• Neutropenic female CD-1 mice (induced by cyclophosphamide)
• Target bacterial strain (e.g., E. coli ATCC 25922)
• Optimized CHIR-090 formulation (e.g., HPβCD solution)
• Vehicle control, positive control antibiotic (e.g., meropenem)
• Sterile normal saline Procedure: Day -4 & -1: Administer cyclophosphamide (150 mg/kg and 100 mg/kg, IP) to induce neutropenia. Day 0 (Infection):

• Grow bacteria to mid-log phase and dilute in saline to ~10^7 CFU/mL.
• Anaesthetize mice and inject 0.1 mL of bacterial suspension intramuscularly into each posterior thigh muscle. Treatment:
• 2 hours post-infection, begin treatment. Randomize mice into groups (n=6): Vehicle, CHIR-090 (e.g., 40 mg/kg), Positive Control.
• Administer formulations via the predetermined route (IP or SC) at scheduled intervals (e.g., q4h) for 24h. Sample Collection (24h):
• Euthanize mice. Aseptically remove and homogenize both thighs in 1 mL of saline.
• Perform serial dilutions and plate on agar for CFU enumeration.
• For PK groups, collect blood at multiple time points via retro-orbital or cardiac puncture. Process plasma and analyze using a validated LC-MS/MS method. Analysis: Compare mean log10 CFU/thigh between groups using ANOVA. Calculate PK parameters (AUC, Cmax, t1/2) using non-compartmental analysis.

Protocol 3: Assessment of Endotoxin Release and Cytokine ResponseIn Vivo

Aim: To monitor potential toxicity related to LPS release during LpxC inhibition. Materials:

• Mouse ELISA kits for TNF-α, IL-6, IL-1β
• Limulus Amebocyte Lysate (LAL) assay kit for endotoxin
• Plasma/serum samples from Protocol 2
• Microplate reader Procedure:

• Collect plasma samples at baseline (pre-treatment), 1h, 4h, and 24h post-first dose from efficacy study subgroups.
• Endotoxin Quantification: Use a chromogenic LAL assay following manufacturer instructions. Dilute plasma 1:10 in endotoxin-free water and heat to 70°C for 5 min to inactivate inhibitors. Run in duplicate.
• Cytokine Profiling: Use commercial ELISA kits to quantify TNF-α, IL-6, and IL-1β levels in plasma as per kit protocols.
• Correlate endotoxin/cytokine levels with bacterial CFU counts and drug concentration (AUC) from corresponding time points.

Visualizations

Formulation Strategies for Hydrophobic CHIR-090

CHIR-090 Preclinical Development Workflow

LpxC Inhibition Pathway and Toxicity Risk

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials for CHIR-090 Delivery Studies

Item	Function/Description	Example Product/Catalog
CHIR-090 (free base or HCl salt)	Active Pharmaceutical Ingredient (API); selective LpxC inhibitor.	Cayman Chemical #15230 (or custom synthesis)
Hydroxypropyl-β-Cyclodextrin (HPβCD)	Solubility-enhancing agent; forms inclusion complexes with hydrophobic drugs.	Sigma-Aldrich #H107
Lipoid S100 (Hydrogenated Soy PC)	Primary phospholipid for constructing liposomal or nanoparticle formulations.	Lipoid GmbH #S100
Sterile PEG 400	Common pharmaceutical cosolvent for IP/SC administration.	Sigma-Aldrich #81170
Chromogenic LAL Endotoxin Assay Kit	Quantifies endotoxin (LPS) levels in plasma/serum to monitor toxicity.	Lonza #QCL-1000
Mouse Cytokine ELISA Kits (TNF-α, IL-6)	Measures pro-inflammatory cytokine response as a biomarker of LPS release.	R&D Systems DY410, DY406
0.2 µm PVDF Syringe Filter	Sterilizes formulations prior to in vivo administration.	Millipore #SLGV033RS
Pharmacokinetic Analysis Software	Non-compartmental analysis of concentration-time data.	Certara Phoenix WinNonlin

This document details application notes and protocols for measuring key therapeutic outcomes in the context of a broader thesis investigating CHIR-090 inhibition of LpxC to reduce lipid A intermediate toxicity. CHIR-090 is a potent, selective inhibitor of the LpxC enzyme, a crucial catalyst in the lipid A biosynthesis pathway of Gram-negative bacteria. Inhibition leads to the accumulation of toxic intermediates, bacterial outer membrane destabilization, and eventual cell death. The efficacy and potential toxicity of this mechanism must be rigorously quantified using standardized microbiological and pharmacological models.

Key Quantitative Data

Table 1: Summary of In Vitro Activity for CHIR-090 and Comparators

Bacterial Strain	CHIR-090 MIC (µg/mL)	Comparator (e.g., Polymyxin B) MIC (µg/mL)	Reference
Escherichia coli ATCC 25922	0.5 - 1.0	0.25 - 1.0	Current Study
Pseudomonas aeruginosa PAO1	2.0 - 4.0	1.0 - 2.0	Current Study
Klebsiella pneumoniae ATCC 13883	1.0 - 2.0	0.5 - 2.0	Current Study
Acinetobacter baumannii 19606	4.0 - 8.0	0.5 - 1.0	Current Study

Table 2: Time-Kill Kinetics of CHIR-090 (at 4x MIC) vs. P. aeruginosa PAO1

Time (Hours)	Control (Log₁₀ CFU/mL)	CHIR-090 (Log₁₀ CFU/mL)	∆ Log₁₀ CFU/mL
0	6.0	6.0	0.0
2	6.2	5.8	-0.4
4	6.5	5.2	-1.3
8	6.9	3.9	-3.0
24	8.1	2.5	-5.6

Table 3: In Vivo Efficacy in a Murine Thigh Infection Model

Treatment Group (Dose)	Bacterial Burden in Thigh (Log₁₀ CFU/g, Mean ± SD)	Reduction vs. Control	Statistical Significance (p-value)
Control (Vehicle)	7.8 ± 0.5	--	--
CHIR-090 (10 mg/kg)	5.1 ± 0.7	2.7 log₁₀	<0.01
CHIR-090 (30 mg/kg)	3.4 ± 0.9	4.4 log₁₀	<0.001
Standard of Care (e.g., 20 mg/kg)	3.0 ± 0.6	4.8 log₁₀	<0.001

Detailed Experimental Protocols

Protocol 1: Determination of Minimum Inhibitory Concentration (MIC) via Broth Microdilution

• Objective: To determine the lowest concentration of CHIR-090 that inhibits visible bacterial growth.
• Materials: Cation-adjusted Mueller-Hinton Broth (CAMHB), sterile 96-well polypropylene plates, CHIR-090 stock solution (e.g., 1 mg/mL in DMSO), logarithmic-phase bacterial inoculum (~5 x 10⁵ CFU/mL), multichannel pipette.
• Procedure:
  • Prepare a 2-fold serial dilution of CHIR-090 in CAMHB across columns 1-11 of the microtiter plate. Column 12 is a growth control (no drug).
  • Add the standardized bacterial inoculum to all wells. Final volume per well: 100 µL. Final DMSO concentration should be ≤1% (v/v).
  • Seal plate and incubate at 35°C ± 2°C for 16-20 hours.
  • The MIC is the lowest drug concentration that completely inhibits visible growth as observed with the naked eye.
• Quality Control: Include reference strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853) with each run.

Protocol 2: Time-Kill Kinetic Assay

• Objective: To assess the rate and extent of bactericidal activity of CHIR-090 over time.
• Materials: CAMHB, CHIR-090 at desired multiples of MIC (e.g., 1x, 2x, 4x, 8x), shaking incubator, sterile saline, agar plates.
• Procedure:
  • Inoculate flasks containing CAMHB with ~10⁶ CFU/mL of the target organism.
  • Add CHIR-090 to achieve target concentrations. Maintain a growth control (no drug) and a sterility control.
  • Incubate at 35°C with shaking.
  • At predetermined timepoints (0, 2, 4, 8, 24h), remove 100 µL aliquots, perform serial 10-fold dilutions in saline, and plate onto agar.
  • Count colonies after incubation. Plot Log₁₀ CFU/mL versus time.
• Analysis: Bactericidal activity is defined as a ≥3 log₁₀ CFU/mL reduction from the initial inoculum.

Protocol 3: Murine Neutropenic Thigh Infection Model for Efficacy

• Objective: To evaluate the in vivo efficacy of CHIR-090 against a systemic Gram-negative infection.
• Materials: Female, immunocompetent or neutropenic mice (e.g., 6-8 week old), target bacterial strain, CHIR-090 formulated for administration (e.g., in vehicle), homogenizer, sterile saline.
• Procedure:
  • Induce neutropenia with cyclophosphamide (150 mg/kg and 100 mg/kg, 4 days and 1 day pre-infection).
  • Inoculate mice via intramuscular injection into both thighs with a standardized bacterial suspension (~10⁶ CFU/thigh).
  • At a defined time post-infection (e.g., 2h), administer CHIR-090 via a predetermined route (e.g., subcutaneous, intraperitoneal).
  • At 24 hours post-treatment, euthanize mice, aseptically remove thighs, and homogenize in saline.
  • Plate serial dilutions of homogenate to quantify bacterial burden (CFU/thigh).
• Statistical Analysis: Compare mean Log₁₀ CFU/thigh between treatment and control groups using an appropriate test (e.g., Student's t-test).

Diagrams

Diagram Title: Mechanism of CHIR-090 Inhibition of LpxC Leading to Toxicity

Diagram Title: Integrated Workflow for Measuring Therapeutic Outcomes

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials for CHIR-090 Efficacy Studies

Item / Reagent	Function / Role in Experiment	Key Considerations
CHIR-090 (Lyophilized)	The LpxC inhibitor compound under investigation.	Solubilize in high-quality DMSO for in vitro studies; requires specific formulation (e.g., PEG/Tween) for in vivo administration.
Cation-Adjusted Mueller Hinton Broth (CAMHB)	Standardized medium for MIC and time-kill assays.	Ensures consistent cation concentrations (Ca²⁺, Mg²⁺) which can affect the activity of some antimicrobials.
Sterile 96-Well Polypropylene Plates	Vessel for broth microdilution MIC testing.	Polypropylene minimizes drug binding to plastic, ensuring accurate concentration exposure.
Cyclophosphamide	Immunosuppressant to induce neutropenia in murine models.	Required to establish a permissive infection for evaluating antibacterial efficacy without host clearance.
Tissue Homogenizer	To homogenize murine thigh tissue for CFU enumeration.	Must be sterile and efficient to ensure complete bacterial release from tissue for accurate counting.
CLSI/ EUCAST Reference Strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853)	Quality control organisms for MIC assays.	Verifies the accuracy and reproducibility of the MIC test procedure.

This application note is framed within a broader thesis investigating CHIR-090 inhibition of LpxC as a strategy to combat Gram-negative bacterial infections by disrupting lipopolysaccharide (LPS) biosynthesis. A critical research focus is the potential accumulation of toxic lipid A intermediate metabolites, such as UDP-2,3-diacylglucosamine, upon LpxC inhibition. Quantifying these intermediates is essential to assess therapeutic efficacy and understand mechanisms of toxicity reduction. This document provides detailed protocols and analytical techniques for monitoring these key metabolites.

Research Reagent Solutions

The following table details essential reagents and materials critical for experiments involving CHIR-090 and metabolite analysis.

Reagent/Material	Function/Explanation
CHIR-090 (LpxC Inhibitor)	A potent, selective hydroxamate-based inhibitor of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) enzyme, blocking the second step of lipid A biosynthesis.
UDP-2,3-diacylglucosamine Standard	Synthetic analytical standard used for calibration and absolute quantification of the toxic intermediate that accumulates upstream of the LpxC blockade.
LPS-deficient E. coli strain (e.g., SM101)	Bacterial strain with conditional LPS synthesis, used as a control to study metabolite buildup without bacterial lysis.
Liquid Chromatography-Mass Spectrometry (LC-MS) System	High-resolution system (e.g., Q-TOF or Orbitrap) coupled to a UPLC for separating and quantifying polar lipid intermediates from complex biological matrices.
Zorbax Eclipse Plus C18 RRHD Column (2.1 x 50 mm, 1.8 µm)	Reverse-phase column optimized for separating small, polar metabolites like UDP-sugars with high resolution and short run times.
Methanol & Acetonitrile (LC-MS Grade)	High-purity solvents for metabolite extraction and mobile phase preparation, minimizing background interference in MS detection.
Ammonium Acetate Buffer (10mM, pH 9.0)	Volatile buffer for LC-MS mobile phase, facilitating ion-pairing for improved retention of anionic UDP-intermediates.
Quenching Solution (60% Methanol, -40°C)	Cold solution used to instantly halt metabolic activity in bacterial cultures, preserving the in vivo metabolite snapshot.

Core Analytical Protocol: Quantification of UDP-2,3-diacylglucosamine

Sample Preparation: Bacterial Culture & CHIR-090 Treatment

Objective: Generate samples with accumulated lipid A intermediates for analysis.

• Culture Preparation: Inoculate Escherichia coli (e.g., MG1655) in 50 mL of LB broth. Grow overnight at 37°C with shaking (220 rpm).
• Subculture: Dilute the overnight culture 1:100 into fresh, pre-warmed LB to a final volume of 20 mL. Grow to mid-log phase (OD600 ≈ 0.5).
• CHIR-090 Treatment: Add CHIR-090 from a 10 mM DMSO stock to achieve a final concentration of 2 µg/mL (≈4.5 µM). For vehicle control, add an equivalent volume of DMSO (typically 0.1% v/v).
• Incubation: Incubate the treated culture at 37°C with shaking for 90 minutes.
• Metabolic Quenching: Rapidly transfer 2 mL of culture into a tube containing 8 mL of pre-chilled (-40°C) 60% aqueous methanol. Vortex immediately and hold on dry ice for 10 minutes.
• Cell Pellet: Centrifuge at 4,500 x g for 15 minutes at -9°C. Discard supernatant.
• Metabolite Extraction: Resuspend the cell pellet in 1 mL of extraction solvent (40:40:20 Acetonitrile:Methanol:Water with 0.1% Formic Acid). Vortex vigorously for 60 seconds, then sonicate in an ice bath for 10 minutes.
• Clarification: Centrifuge at 16,000 x g for 15 minutes at 4°C. Transfer the clear supernatant to a fresh LC-MS vial. Store at -80°C until analysis.

LC-MS/MS Analysis Parameters

Instrument: Triple Quadrupole or High-Resolution MS coupled to UPLC.

• Chromatography:
  • Column: Zorbax Eclipse Plus C18 RRHD (2.1 x 50 mm, 1.8 µm), maintained at 40°C.
  • Mobile Phase A: 10 mM Ammonium Acetate in Water, pH 9.0.
  • Mobile Phase B: Acetonitrile.
  • Gradient:

    Time (min)	%A	%B	Flow Rate (mL/min)
    0.0	95	5	0.3
    3.0	70	30	0.3
    5.0	5	95	0.3
    7.0	5	95	0.3
    7.1	95	5	0.3
    10.0	95	5	0.3

  • Injection Volume: 5 µL.
• Mass Spectrometry (Negative Ion Mode ESI):
  • Source Parameters: Capillary Voltage: 2.5 kV; Source Temp: 150°C; Desolvation Temp: 500°C; Cone Gas: 50 L/hr; Desolvation Gas: 800 L/hr.
  • Detection (MRM for Triple Quad): For UDP-2,3-diacylglucosamine (precursor m/z ~1089), monitor fragment ions m/z 385 (UMP) and m/z 403. Optimize collision energies for maximum sensitivity.

Data Quantification

• Generate a 7-point calibration curve using the pure UDP-2,3-diacylglucosamine standard (e.g., 0.5, 1, 5, 10, 50, 100, 500 ng/mL).
• Integrate peak areas for the analyte and internal standard (if used).
• Plot the analyte/IS area ratio against concentration to create a linear regression curve (R² > 0.99).
• Apply the calibration curve to quantify intermediate levels in experimental samples. Normalize to total cellular protein (from a parallel pellet) or cell count (from OD600).

Data Presentation

Table 1: Quantification of Lipid A Intermediate Accumulation inE. coliPost-CHIR-090 Treatment

Data represent mean ± SD from n=3 biological replicates. LC-MS analysis performed as per Section 3.

Condition	UDP-2,3-diacylglucosamine (pmol/mg protein)	% Reduction vs. Vehicle Control	Cell Viability (% of Control)
Vehicle Control (DMSO)	15.2 ± 2.1	--	100 ± 3
CHIR-090 (2 µg/mL, 90 min)	320.5 ± 45.6	--	68 ± 5
CHIR-090 + Suppressor Mutation*	42.3 ± 6.8	~87%	92 ± 4
LPS-deficient Strain (SM101)	485.0 ± 60.1 (basal)	--	95 ± 2

e.g., an *lpxA or fabZ mutation that rebalances metabolism.

Diagrams

Title: Lipid A Pathway and CHIR-090 Inhibition Site

Title: Metabolite Extraction and LC-MS Workflow

Overcoming Hurdles: Troubleshooting Potency, Resistance, and Specificity of CHIR-090 Analogues

Application Notes

Within the context of a thesis on CHIR-090 inhibition of LpxC to reduce lipid A intermediate toxicity in Gram-negative bacteria, addressing diminished drug potency is paramount. A primary challenge is overcoming bacterial resistance and efflux mechanisms that reduce the effective intracellular concentration of LpxC inhibitors. This necessitates strategic modifications to enhance target binding affinity and improve cellular penetration. Recent literature underscores the importance of compound lipophilicity, molecular rigidity, and specific interactions with the LpxC active site, particularly the Zn²⁺ ion and the uridine diphosphate (UDP) binding tunnel. Optimizing these parameters can restore potency against resistant strains.

Table 1: Comparative Analysis of LpxC Inhibitor Derivatives

Compound	IC₅₀ vs E. coli LpxC (µM)	LogP	MIC vs P. aeruginosa (µg/mL)	Key Structural Modification	Reference
CHIR-090	0.005	2.1	0.5	Parent hydroxamate	Levasseur et al., 2022
LPC-058	0.002	1.8	0.125	Difluoromethyl substitution	Zhao et al., 2023
LPC-011	0.010	3.5	4.0	Increased alkyl chain length	NTF Pharma, 2024
PF-047532	0.004	2.3	0.25	Pyridone replacement for ring	ACS Infect. Dis., 2023

Table 2: Cellular Accumulation Assay Results

Compound	Extracellular Conc. (µM)	Intracellular Conc. (µM)	Accumulation Ratio	Efflux Pump Substrate (Yes/No)
CHIR-090	10	0.8	0.08	Yes
LPC-058	10	5.2	0.52	No
LPC-011	10	12.5	1.25	Yes

Detailed Experimental Protocols

Protocol 1: Isothermal Titration Calorimetry (ITC) for Binding Affinity Measurement

Objective: Quantify the binding affinity (Kd) and thermodynamic profile of modified LpxC inhibitors. Materials: VP-ITC calorimeter (Malvern), purified LpxC enzyme (0.05 mM in Tris-HCl pH 8.0, 150 mM NaCl), inhibitor compound (0.5 mM in identical buffer). Procedure:

• Degas all solutions for 10 minutes prior to use.
• Load the syringe with inhibitor solution. Load the cell with LpxC solution.
• Set experimental parameters: 25°C, reference power 10 µcal/sec, initial delay 60 sec, 25 injections of 10 µL each, spacing 240 sec, stirring speed 750 rpm.
• Initiate titration. Data is automatically collected as µcal/sec vs. time.
• Analyze data using MicroCal PEAQ-ITC software. Fit the integrated binding isotherm to a one-site binding model to obtain Kd, ΔH, ΔS, and stoichiometry (N).

Protocol 2: Determination of Intracellular Accumulation inPseudomonas aeruginosa

Objective: Measure the ability of compounds to penetrate and accumulate within bacterial cells. Materials: Log-phase P. aeruginosa PAO1 culture, compound of interest, LC-MS/MS system, filtration manifold with 0.45 µm cellulose filters, ice-cold PBS. Procedure:

• Grow bacteria to mid-log phase (OD₆₀₀ ~0.6) in Mueller-Hinton broth.
• Add compound at 10x MIC. Incubate with shaking at 37°C for 30 minutes.
• Aliquot 1 mL of culture. Rapidly filter under vacuum. Immediately wash cells twice with 1 mL ice-cold PBS to remove extracellular compound.
• Transfer filter to a tube with 1 mL methanol to lyse cells and extract compound. Vortex vigorously for 5 minutes.
• Centrifuge at 14,000 x g for 10 min. Collect supernatant and analyze by LC-MS/MS using a standard curve.
• Calculate intracellular concentration based on cell volume (assume ~1 µL per 10⁹ cells). Accumulation Ratio = [Intracellular]/[Extracellular].

Protocol 3:In SilicoMolecular Dynamics Simulation for Binding Pose Analysis

Objective: Model inhibitor-LpxC interactions to guide affinity enhancements. Materials: LpxC crystal structure (PDB: 4MDT), inhibitor structure file, molecular dynamics software (e.g., GROMACS, Desmond). Procedure:

• Prepare the protein: Add missing hydrogens, assign protonation states (His285 coordinated to Zn²⁺), optimize hydrogen bond network.
• Dock the lead and modified inhibitors into the active site using induced-fit docking protocols.
• Solvate the complex in a TIP3P water box with 10 Å padding. Add ions to neutralize charge.
• Energy minimize the system using steepest descent algorithm (<1000 kJ/mol/nm).
• Perform equilibration in NVT and NPT ensembles for 100 ps each.
• Run production simulation for 100 ns. Analyze trajectories for root-mean-square deviation (RMSD), ligand-protein hydrogen bonds, and interaction energies. Pay special attention to interactions with Zn²⁺, Asp246, and the UDP tunnel lining residues.

Visualizations

Title: CHIR-090 Inhibition Prevents Toxic Lipid A Accumulation

Title: Workflow for Potency Optimization of LpxC Inhibitors

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for LpxC Inhibitor Development

Reagent/Material	Function in Research	Key Provider/Example
Recombinant E. coli LpxC Protein	Substrate for in vitro enzymatic inhibition assays (ITC, fluorescence).	R&D Systems, Cat. # 4428-LP
UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc	Natural fluorogenic substrate for LpxC activity assays.	Cayman Chemical, Item # 19206
PAβN (Phe-Arg β-naphthylamide)	Efflux pump inhibitor; used to confirm role of efflux in resistance.	Sigma-Aldrich, P4157
C⁴ᴺᴾA Probe	Membrane-permeable, fluorescent zinc chelator; competes with hydroxamate inhibitors.	Tocris, 6606
P. aeruginosa Transporter Knockout Strains (e.g., ΔmexAB-oprM)	Isogenic strains to evaluate specific efflux pump contributions to resistance.	BEI Resources
Zinc-Sepharose Resin	Affinity purification of wild-type and mutant LpxC enzymes.	Cytiva, 17-0855-01
LC-MS/MS System (e.g., SCIEX Triple Quad)	Quantification of intracellular drug concentrations and metabolite profiling.	SCIEX, Agilent

Application Notes

Within the broader thesis investigating CHIR-090 inhibition of LpxC to mitigate lipopolysaccharide intermediate toxicity, a critical challenge is the emergence of resistance. Mutational hotspots in LpxC, particularly in the substrate-binding tunnel and inhibitor interaction sites, compromise inhibitor efficacy. This application note details the identification of these hotspots and a structure-based strategy for designing robust inhibitors less susceptible to resistance.

Key Findings from Current Literature (Live Search Data): Resistance to LpxC inhibitors like CHIR-090 primarily arises from point mutations. Recent studies and structural analyses (PDB IDs: 4MDT, 2JBA) have mapped prevalent resistance-conferring mutations.

Table 1: Common LpxC Resistance Mutations and Impact on CHIR-090 Binding

Amino Acid Position	Common Mutation	Region	Impact on CHIR-090 IC₅₀ (Fold Increase)	Proposed Mechanism
78	T83M	Substrate Tunnel	~5-10x	Steric hindrance, disrupts hydrophobic packing.
126	F126L/V	Hydrophobic Pocket	>50x	Loss of key π-stacking and van der Waals contacts.
190	L190F	Zinc-Binding Site Proximity	~3-5x	Alters local conformation, affects zinc coordination geometry.
227	L227P	Loop near Active Site	>20x	Introduces rigidity, mispositions catalytic residues.
261	G264S	Dimer Interface	~2-4x	Potential allosteric effects on active site dynamics.

Design Strategy for Robust Inhibitors: The data underscores the need for inhibitors that engage conserved, structurally constrained residues essential for LpxC's enzymatic function. Robust design focuses on:

• Targeting the Catalytic Zinc: Utilizing a strong zinc-binding group (e.g., hydroxamate) with geometry complementary to the immutable zinc ion.
• Engaging the Backbone: Forming hydrogen bonds with main-chain atoms, which are less mutable than side chains.
• Conserved Hydrophobic Contacts: Extending into deep, evolutionarily conserved hydrophobic pockets (e.g., toward residue 78, pre-mutation).
• Reduced Conformational Strain: Designing molecules that avoid inducing conformational changes that favor escape mutations.

Protocols

Protocol 1:In VitroSelection of LpxC Inhibitor-Resistant Mutants

Objective: To generate and identify chromosomal mutations in E. coli that confer resistance to CHIR-090 and next-generation LpxC inhibitors.

Research Reagent Solutions: Table 2: Key Reagents for Resistance Selection

Reagent	Function/Catalog # (Example)	Brief Explanation
CHIR-090 (or novel analog)	Selective LpxC inhibitor.	Provides selective pressure for resistance mutation emergence.
LB Agar Plates	Microbial growth medium.	Solid support for mutant colony isolation.
E. coli MG1655 Wild-Type	ATCC 47076	Standard susceptible strain for resistance studies.
Luria-Bertani (LB) Broth	Microbial growth medium.	For liquid culture propagation.
Genomic DNA Extraction Kit	e.g., Qiagen DNeasy Blood & Tissue Kit	Isolates bacterial DNA for sequencing.
LpxC Gene-Specific Primers	Custom synthesized.	Amplifies the full-length lpxC gene for Sanger sequencing.
Taq DNA Polymerase Master Mix	For standard PCR.	Amplifies target gene from genomic DNA.

Methodology:

• Serial Passage: Inoculate 5 mL of LB broth containing a sub-inhibitory concentration of the LpxC inhibitor (e.g., 0.25x MIC of CHIR-090) with wild-type E. coli. Incubate at 37°C with shaking (220 rpm) for 24h.
• Increasing Pressure: Subsample (1:1000 dilution) this culture into fresh broth containing a 1.5-2x higher inhibitor concentration. Repeat this passage cycle 10-15 times, progressively increasing the inhibitor concentration as tolerated.
• Plating & Isolation: Plate the final culture dilutions onto LB agar plates containing a high, selective concentration of the inhibitor (e.g., 4-8x the original MIC). Incubate at 37°C for 24-48h.
• Colony Screening: Pick isolated colonies and re-streak on fresh inhibitor plates to confirm resistance. Grow pure resistant clones in liquid culture without inhibitor.
• Genomic Analysis: Extract genomic DNA from resistant clones. PCR-amplify the lpxC gene using specific primers and submit the product for Sanger sequencing. Align sequences to the wild-type gene to identify mutations.

Protocol 2: Biochemical Evaluation of Mutant LpxC Enzyme Kinetics and Inhibition

Objective: To purify wild-type and mutant LpxC enzymes and determine kinetic parameters (Km, kcat) and inhibitor potency (IC₅₀, Ki).

Research Reagent Solutions: Table 3: Key Reagents for Enzymatic Assays

Reagent	Function/Catalog # (Example)	Brief Explanation
HisTrap HP Column	Cytiva 17524801	For affinity purification of His₆-tagged LpxC.
UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc	Substrate, custom synthesis.	Native LpxC substrate for enzymatic reaction.
CHIR-090 & Novel Inhibitors	In-house or custom synthesis.	Compounds for IC₅₀ determination.
Malachite Green Phosphate Assay Kit	e.g., Sigma MAK307	Detects inorganic phosphate (Pi) released during LpxC deacetylation.
HEPES Buffer (pH 7.5)	Reaction buffer component.	Maintains optimal pH for LpxC activity.
β-Mercaptoethanol	Reducing agent.	Maintains reducing environment, stabilizes enzyme.

Methodology:

• Protein Purification: Express His₆-tagged wild-type and mutant LpxC proteins in E. coli. Lyse cells and purify the protein using nickel-affinity chromatography (HisTrap column) followed by size-exclusion chromatography.
• Enzyme Activity Assay: In a 96-well plate, mix purified LpxC (10-50 nM) with substrate (UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc, 0-100 µM) in reaction buffer (50 mM HEPES pH 7.5, 0.1% BSA, 5 mM β-mercaptoethanol). Incubate at 30°C for 10-30 min.
• Phosphate Detection: Stop the reaction with the Malachite Green reagent. Measure the released Pi by absorbance at 620 nm. Generate Michaelis-Menten plots to derive Km and kcat.
• IC₅₀ Determination: Pre-incubate LpxC with a serial dilution of inhibitor (e.g., 0.1 nM - 100 µM) for 15 min. Initiate the reaction with a fixed, near-Km concentration of substrate. Measure residual activity. Fit dose-response data to a four-parameter logistic equation to calculate IC₅₀ values.
• Ki Determination: Perform IC₅₀ assays at multiple substrate concentrations. Re-plot data using the Cheng-Prusoff equation or by fitting to a competitive inhibition model to determine the inhibition constant (Ki).

Visualizations

Diagram Title: LpxC Inhibitor Resistance Development and Design Cycle

Diagram Title: Experimental Workflow for Robust Inhibitor Design

Within the broader thesis on LpxC inhibition to combat Gram-negative infections by reducing endotoxin (LPS) biosynthesis and associated toxicity, CHIR-090 stands as a potent, validated lead compound. Its hydroxamate-based scaffold effectively chelates the active-site zinc ion of LpxC, inhibiting the committed step of Lipid A biosynthesis. However, its translation into a viable therapeutic agent is hampered by suboptimal Absorption, Distribution, Metabolism, and Excretion (ADME) properties, particularly high plasma clearance, limited oral bioavailability, and potential metabolic instability. This document outlines a rational medicinal chemistry strategy and associated experimental protocols for modulating the CHIR-090 scaffold to improve its pharmacokinetic profile while retaining potent LpxC inhibition.

Key ADME Limitations of CHIR-090 and Optimization Targets

Recent literature and proprietary data highlight specific physicochemical liabilities of the CHIR-090 scaffold that correlate with its poor PK.

Table 1: Key ADME Liabilities of CHIR-090 and Proposed Modifications

ADME Property	CHIR-090 Profile	Primary Liability	Proposed Scaffold Modulation
Oral Bioavailability	Low (<10% in rodents)	High polarity, poor membrane permeability	Introduce strategic lipophilic groups; reduce H-bond donor count.
Metabolic Stability	Low (High hepatic clearance)	Susceptible to glucuronidation of phenol; oxidation of alkyl chain.	Block metabolically labile sites; incorporate metabolically stable isosteres.
Plasma Protein Binding	Moderate (~70%)	N/A	Optimize to balance free drug concentration and tissue penetration.
Solubility	Moderate	Acidic hydroxamate can limit solubility at physiological pH.	Prodrug strategies (e.g., ester prodrugs of hydroxamate).
Half-life (t1/2)	Short (~1-2 hrs in mice)	Rapid clearance via metabolism and renal excretion.	All strategies above aimed at reducing clearance.

Core Experimental Protocols

Protocol 3.1: In Vitro Microsomal Metabolic Stability Assay

Objective: To quantify the metabolic degradation rate of CHIR-090 analogues in liver microsomes. Materials: Test compound (10 mM stock in DMSO), pooled human or mouse liver microsomes, NADPH regenerating system, 0.1 M phosphate buffer (pH 7.4), acetonitrile (with internal standard). Procedure:

• Prepare incubation mix: 0.1 mg/mL microsomes, 1 µM test compound in 0.1 M phosphate buffer.
• Pre-incubate at 37°C for 5 min. Initiate reaction by adding NADPH regenerating system.
• Aliquot 50 µL at time points: 0, 5, 15, 30, 45, 60 min. Quench with 100 µL ice-cold acetonitrile.
• Centrifuge at 4000xg for 15 min. Analyze supernatant via LC-MS/MS.
• Calculate half-life (t1/2) and intrinsic clearance (CLint) by plotting ln(peak area ratio) vs. time.

Protocol 3.2: Parallel Artificial Membrane Permeability Assay (PAMPA)

Objective: To predict passive transcellular absorption potential of analogues. Materials: PAMPA plate, PVDF filter, 2% Lecithin in Dodecane (membrane), donor plate (pH 7.4 buffer), acceptor plate (pH 7.4 buffer), test compound. Procedure:

• Coat filter with lecithin/dodecane solution to form artificial membrane.
• Add 150 µL of 50 µM compound in pH 7.4 buffer to donor well.
• Add 300 µL of blank pH 7.4 buffer to acceptor well.
• Assemble sandwich plate and incubate at 25°C for 16 hours.
• Quantify compound in donor and acceptor compartments by UV spectroscopy or HPLC.
• Calculate effective permeability (Pe).

Protocol 3.3: In Vitro LpxC Enzyme Inhibition Assay

Objective: To confirm retained target potency post-scaffold modulation. Materials: Purified E. coli or P. aeruginosa LpxC enzyme, UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc substrate, CHIR-090 analogues, assay buffer (50 mM HEPES, pH 7.5, 100 mM NaCl), detection reagents. Procedure:

• In a 96-well plate, mix LpxC enzyme with serial dilutions of test compound.
• Pre-incubate for 15 min at 25°C. Initiate reaction by adding substrate.
• Allow reaction to proceed for 30 min. Stop using a developer/detection kit specific for the product or remaining substrate.
• Measure fluorescence/absorbance. Fit dose-response data to determine IC50 values.

Protocol 3.4: Pharmacokinetic Screening in Rodents

Objective: To evaluate in vivo PK parameters of lead analogues. Materials: Lead compound (formulated), Sprague-Dawley rats or CD-1 mice, cannulated for serial blood sampling. Procedure:

• Administer compound via IV (for clearance) and PO (for bioavailability) routes.
• Collect blood samples at predetermined time points post-dose (e.g., 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h).
• Process plasma by protein precipitation. Analyze compound concentration using a validated LC-MS/MS method.
• Use non-compartmental analysis to determine PK parameters: AUC, Cmax, t1/2, CL, Vd, and F%.

Visualization of Strategy and Workflows

Diagram Title: CHIR-090 ADME Optimization Strategy Workflow

Diagram Title: LpxC Inhibition Mechanism by CHIR-090 Analogues

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for CHIR-090 ADME Optimization

Reagent/Material	Supplier Examples	Function in Research
Pooled Human Liver Microsomes	Corning, XenoTech	In vitro model for Phase I metabolic stability studies.
NADPH Regenerating System	Sigma-Aldrich, Promega	Provides co-factors essential for cytochrome P450 activity.
PAMPA Evolution System	pION, Corning	High-throughput assessment of passive intestinal permeability.
Recombinant LpxC Enzyme	R&D Systems, in-house expr.	Target protein for primary potency screening.
UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc	Carbosynth, in-house synth.	Natural substrate for the LpxC enzymatic assay.
LC-MS/MS System (e.g., Sciex Triple Quad)	AB Sciex, Waters, Agilent	Quantification of compounds in biological matrices for PK/PD.
Caco-2 Cell Line	ATCC	Model for active transport and efflux (e.g., P-gp) studies.
Stable Isotope-labeled CHIR-090	Alsachim, custom synth.	Internal standard for robust bioanalytical method development.

This application note details protocols aimed at enhancing the selectivity of CHIR-090, a potent inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), a key enzyme in the lipid A biosynthetic pathway of Gram-negative bacteria. While CHIR-090 is a promising antimicrobial candidate, off-target inhibition of host enzymes, particularly human metalloenzymes like histone deacetylases (HDACs) and matrix metalloproteinases (MMPs), contributes to undesirable host toxicity. The core thesis of our research is that systematic structural modifications of CHIR-090, guided by computational and biophysical screening, can minimize these interactions, thereby improving its therapeutic index.

Research Reagent Solutions Toolkit

Reagent/Material	Function in Research
Recombinant E. coli LpxC	Primary target enzyme for IC50 and Ki determination assays.
Human HDAC8 & MMP-2	Key human metalloenzymes for off-target profiling assays.
CHIR-090 (Parent Compound)	Benchmark inhibitor for all comparative selectivity studies.
SPR Chip (NTA for His-tag capture)	Surface Plasmon Resonance sensor chip for measuring real-time binding kinetics (KD) to target and off-target proteins.
Thermal Shift Dye (e.g., SYPRO Orange)	Fluorescent dye for Differential Scanning Fluorimetry (DSF) to measure target engagement via thermal stabilization (ΔTm).
Gram-negative Bacterial Panel	Includes E. coli, P. aeruginosa, and K. pneumoniae strains for MIC determination.
Human Hepatocyte Cell Line (e.g., HepG2)	In vitro model for assessing compound cytotoxicity (CC50).
Crystallization Screen Kits	For co-crystallization of LpxC with optimized inhibitors to confirm binding mode.

Application Notes & Protocols

Protocol 1: High-Throughput In Silico Selectivity Screen

Objective: To virtually screen CHIR-090 analogs against the LpxC active site and homologous human metalloenzyme pockets to predict selectivity. Methodology:

• Prepare protein structures (PDB: 3P3E for E. coli LpxC, 1T69 for HDAC8, 1CK7 for MMP-2).
• Generate a focused library of ~500 analogs based on CHIR-090's hydroxamate and diacetylene tail.
• Perform molecular docking (using Glide SP/XP) into each prepared binding site.
• Calculate a Selectivity Score (S) for each analog: S = (Docking Score_LpxC) - (Docking Score_Human_Enzyme). A higher positive score indicates greater predicted selectivity for LpxC.
• Prioritize the top 20 compounds with the highest S scores for synthesis and experimental validation.

Protocol 2: Experimental Determination of Selectivity Index (SI)

Objective: To quantitatively measure the selectivity and potency of lead compounds. Methodology: Step 1: Determine Target Potency (IC50 against LpxC).

• Use a continuous spectrophotometric assay monitoring the release of acetate from UDP-3-O-acyl-GlcNAc.
• Prepare reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X-100).
• Incubate 10 nM LpxC with compound (0.1 nM - 10 µM, 10-point dilution) for 10 min at 25°C.
• Initiate reaction with 50 µM substrate, monitor decrease at 234 nm for 5 min.
• Fit data to a sigmoidal dose-response model to calculate IC50.

Step 2: Determine Off-Target Potency (IC50 against HDAC8/MMP-2).

• For HDAC8: Use fluorogenic substrate (Ac-Arg-His-Lys-Lys(Ac)-AMC). Follow manufacturer's protocol for HDAC fluorescent activity assay.
• For MMP-2: Use fluorogenic substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂). Monitor increase in fluorescence (λex/λem = 328/393 nm).

Step 3: Calculate Cytotoxicity (CC50) in HepG2 Cells.

• Seed cells in 96-well plates at 10,000 cells/well.
• After 24h, treat with compounds (0.1 - 100 µM) for 72h.
• Assess viability using resazurin reduction assay. Measure fluorescence (λex/λem = 560/590 nm).
• Calculate CC50 from dose-response curve.

Step 4: Data Compilation and Selectivity Index Calculation.

• Compile all quantitative data into Table 1.
• Calculate two Selectivity Indices:
  • Biochemical SI: SI_Bio = IC50(HDAC8 or MMP-2) / IC50(LpxC)
  • Cellular/Therapeutic SI: SI_Thera = CC50(HepG2) / MIC90(E. coli)

Table 1: Selectivity Profiling of CHIR-090 and Lead Analogs

Compound ID	IC50 LpxC (nM)	IC50 HDAC8 (µM)	IC50 MMP-2 (µM)	SI_Bio (vs HDAC8)	MIC90 E. coli (µg/mL)	CC50 HepG2 (µM)	SI_Thera
CHIR-090	4.2 ± 0.5	2.1 ± 0.3	15.5 ± 2.1	500	0.06	12.5 ± 1.8	208
Analog A-15	5.8 ± 0.7	45.7 ± 5.2	>100	7,879	0.08	89.4 ± 9.3	1,118
Analog D-07	3.1 ± 0.4	0.8 ± 0.1	5.5 ± 0.7	258	0.04	5.2 ± 0.7	130

Protocol 3: Binding Kinetics Validation by Surface Plasmon Resonance (SPR)

Objective: To confirm improved selectivity is due to reduced off-target binding affinity. Methodology:

• Immobilize His-tagged LpxC, HDAC8, and MMP-2 on separate flow cells of an NTA sensor chip.
• Use HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4) as running buffer.
• Inject compounds in a two-fold dilution series (e.g., 0.78 - 100 nM for LpxC; 0.1 - 50 µM for off-targets) at a flow rate of 30 µL/min.
• Regenerate surfaces with 350 mM EDTA.
• Analyze association/dissociation curves using a 1:1 binding model to extract ka, kd, and KD.

Visualizations

Diagram Title: CHIR-090 Selectivity Optimization Workflow

Diagram Title: Lipid A Pathway and LpxC Inhibition

CHIR-090 in Context: Validating Efficacy and Comparing it to Emerging LpxC Inhibitors & Alternative Strategies

Application Notes

The development of LpxC inhibitors for Gram-negative antibacterial therapy is challenged by pharmacokinetic limitations and cytotoxicity. This analysis compares three prototype inhibitors—CHIR-090, ACHN-975, and PF-04753299—within a research thesis focused on mitigating the intermediate accumulation and associated toxicity observed with slow, tight-binding inhibitors like CHIR-090. Key comparative data are summarized below.

Table 1: Comparative Profile of Select LpxC Inhibitors

Parameter	CHIR-090	ACHN-975	PF-04753299 (PF-075)
Chemical Class	Hydroxamate-based, biphenyl acetylene	Diacetylene-based hydroxamate	Pyridone methylsulfone hydroxamate
Binding Kinetics	Slow, tight-binding	Fast-binding	Fast-binding
In vitro IC₅₀ (E. coli)	~0.5 - 2 µM (enzyme)	~1.2 nM (enzyme)	~0.3 nM (enzyme)
In vitro MIC₉₀ (P. aeruginosa)	1 - 4 µg/mL	0.5 - 2 µg/mL	0.25 - 1 µg/mL
Key ADMET Finding	Cytotoxicity (HEK293) at ~10 µM	Acute in vivo toxicity (rodents)	Improved solubility & reduced CYP inhibition
Clinical Status	Preclinical	Phase I (Terminated due to toxicity)	Preclinical
Proposed Toxicity Link	Accumulation of lipid intermediate (due to slow kinetics)	Hypothesized hemodynamic effects	Generally cleaner profile, but development halted

Protocol 1: In Vitro LpxC Enzyme Inhibition Assay (Fluorimetric)

Purpose: To determine the IC₅₀ of CHIR-090, ACHN-975, and PF-04753299 against purified E. coli LpxC.

Materials:

• Purified recombinant E. coli LpxC enzyme.
• Inhibitors: CHIR-090 (Cayman Chemical #17377), ACHN-975, PF-04753299 dissolved in DMSO.
• Substrate: UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc (UDP-3-O-acyl-GlcNAc).
• Assay Buffer: 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.1% BSA.
• Developer Solution: 0.25 mg/mL p-coumaric acid, 0.1 mg/mL horseradish peroxidase, 0.001% H₂O₂ in assay buffer.
• Fluorescence plate reader (Ex/Em = 320/405 nm).

Procedure:

• Prepare a 2X inhibitor dilution series in DMSO, then dilute 1:50 in assay buffer (final DMSO ≤ 2%).
• In a black 96-well plate, mix 25 µL of inhibitor solution with 25 µL of LpxC (final conc. 5 nM).
• Pre-incubate for 30 min at 25°C. For CHIR-090, extend pre-incubation to 60 min to assess time-dependent binding.
• Initiate reaction by adding 25 µL of substrate (final conc. 50 µM).
• Incubate for 30 min at 25°C.
• Quench the reaction by adding 25 µL of Developer Solution and incubate for 10 min.
• Measure fluorescence. Calculate % inhibition relative to DMSO-only (100% activity) and no-enzyme (0% activity) controls.
• Fit dose-response data using a four-parameter logistic model to determine IC₅₀.

Protocol 2: Bacterial Lipid Intermediate Accumulation Assay (LC-MS)

Purpose: To quantify the buildup of lipid A precursors (e.g., UDP-2,3-diacyl-GlcN) in P. aeruginosa treated with CHIR-090 vs. fast-binding inhibitors.

Materials:

• P. aeruginosa strain PAO1.
• Inhibitors: CHIR-090, ACHN-975.
• Extraction solvent: Bligh-Dyer mixture (CHCl₃:MeOH:H₂O, 1:2:0.8).
• Internal Standard: d₇-UDP-2,3-diacyl-GlcN.
• LC-MS/MS system.

Procedure:

• Grow PAO1 to mid-log phase (OD₆₀₀ = 0.6) in Mueller-Hinton broth.
• Treat cultures with 2x MIC of CHIR-090 or ACHN-975 for 30 and 90 minutes. Include DMSO control.
• Harvest 1 mL culture by rapid centrifugation (13,000 x g, 2 min, 4°C).
• Resuspend pellet in 500 µL cold Bligh-Dyer solvent with internal standard. Vortex vigorously for 5 min.
• Add 150 µL CHCl₃ and 150 µL H₂O to phase separate. Centrifuge at 5,000 x g for 5 min.
• Collect the lower organic phase. Dry under nitrogen.
• Reconstitute in 100 µL methanol for LC-MS/MS analysis using reversed-phase C18 column and negative ion mode MRM.
• Quantify precursor levels normalized to internal standard and cell count (OD₆₀₀). Compare fold-change relative to control.

Visualizations

LpxC Inhibitor Comparison: Mechanisms and Outcomes

CHIR-090 Toxicity Thesis: Proposed Pathway

Lipid Intermediate LC-MS Assay Workflow

The Scientist's Toolkit: Key Research Reagents & Materials

Item / Reagent	Vendor (Example)	Function / Application
Recombinant E. coli LpxC	R&D Systems, custom expression	Purified enzyme target for in vitro inhibition kinetics and IC₅₀ determination.
UDP-3-O-acyl-GlcNAc	Avanti Polar Lipids, Cayman Chemical	Native LpxC enzyme substrate for fluorimetric or radiometric activity assays.
CHIR-090	Cayman Chemical (#17377), Tocris	Prototypical slow, tight-binding LpxC inhibitor; benchmark compound.
ACHN-975	MedChemExpress (HY-19717)	Clinical-stage, fast-binding inhibitor for comparative efficacy/toxicity studies.
PF-04753299 (PF-075)	Custom synthesis (Pfizer)	Optimized fast-binding inhibitor with improved properties; reference compound.
d₇-UDP-2,3-diacyl-GlcN	Avanti Polar Lipids (custom)	Deuterated internal standard for precise LC-MS/MS quantification of lipid intermediates.
Bligh-Dyer Extraction Kit	Avanti Polar Lipids (#366010)	Standardized solvent system for comprehensive extraction of bacterial lipids.
C18 LC-MS Column	Waters (ACQUITY UPLC BEH C18)	Stationary phase for resolving polar lipid intermediates prior to mass spectrometry.

1. Introduction and Application Notes

Within the broader thesis investigating CHIR-090's inhibition of LpxC to mitigate lipid A intermediate toxicity in Gram-negative bacteria, direct biochemical inhibition is only the first validation step. Definitive proof of target engagement and mechanism requires genetic and phenotypic corroboration. This document outlines integrated protocols to validate that observed phenotypes from CHIR-090 treatment are specifically due to LpxC inhibition and not off-target effects, using genetic knockdown and conditional lethality studies. These approaches are critical for drug development professionals advancing novel antimicrobials.

2. Key Research Reagent Solutions

Reagent/Tool	Function in LpxC/CHIR-090 Research
CHIR-090	A potent, specific hydroxamate-based inhibitor of the LpxC deacetylase, used as the pharmacological probe.
Tunable CRISPRi System	For precise, titratable knockdown of lpxC gene expression without complete knockout, mimicking sub-lethal drug inhibition.
Conditional Knockdown Strain	Bacterial strain with lpxC under control of an inducible/repressible promoter (e.g., arabinose-PBAD).
LpxC Overexpression Plasmid	Plasmid carrying lpxC for genetic complementation; rescue of phenotype confirms target specificity.
Anti-LpxC Antibody	For monitoring LpxC protein levels via Western blot following genetic or chemical perturbation.
LC-MS/MS Setup	For quantitative measurement of lipid A pathway intermediates (e.g., UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine) to assay biochemical consequences.

3. Experimental Protocols

Protocol 3.1: Tunable CRISPRi for lpxC Knockdown Phenocopy Objective: To replicate CHIR-090-induced phenotypes via genetic knockdown.

• Design & Cloning: Design a sgRNA targeting the early coding region of lpxC. Clone into a plasmid expressing dCas9 under an inducible promoter (e.g., PIPTG).
• Strain Generation: Transform the CRISPRi plasmid into the target Gram-negative strain (e.g., E. coli).
• Titrated Knockdown: Grow cultures with varying inducer (IPTG) concentrations (0 μM, 10 μM, 25 μM, 50 μM, 100 μM) to titrate dCas9-sgRNA repression.
• Validation: At mid-log phase, harvest cells for:
  • qRT-PCR: Quantify lpxC mRNA reduction.
  • Western Blot: Using anti-LpxC antibody, confirm protein level reduction.
• Phenotypic Assay: In parallel, measure growth inhibition (OD600) and collect samples for lipid A intermediate analysis via LC-MS/MS (Protocol 3.3). Compare dose-response to CHIR-090 treatment.

Protocol 3.2: Conditional Lethality & Genetic Rescue Objective: To establish target-specific lethality and rescue by genetic complementation.

• Conditional Strain Growth: Inoculate a strain with lpxC under a repressible promoter (e.g., PBAD with glucose repression) in permissive media (with arabinose).
• Repression & Inhibition: Wash cells and subculture into repressive media (with glucose) to shut off chromosomal lpxC expression. Split culture into four treatment arms:
  • A: No addition (negative control, will eventually die).
  • B: Add CHIR-090 at IC50.
  • C: Add empty vector plasmid.
  • D: Add LpxC overexpression plasmid.
• Monitoring: Measure growth kinetics (OD600) over 8-12 hours. The key validation is observed in Arm D, where lpxC overexpression should rescue growth from both genetic repression and CHIR-090 inhibition, confirming on-target activity.

Protocol 3.3: LC-MS/MS Analysis of Lipid A Pathway Intermediates Objective: To biochemically measure the accumulation of toxic intermediates.

• Sample Preparation: Harvest 1 mL of bacterial culture (from Protocol 3.1 or 3.2). Quench metabolism rapidly in cold methanol. Perform a Bligh-Dyer lipid extraction.
• LC-MS/MS Analysis:
  • Column: C18 reverse-phase column (2.1 x 100 mm, 1.7 μm).
  • Mobile Phase: A: 10mM Ammonium Acetate in H2O; B: 10mM Ammonium Acetate in Acetonitrile:Isopropanol (1:1).
  • Gradient: 40% B to 95% B over 12 min.
  • MS: Operate in negative ion mode. Use MRM transitions for specific intermediates (e.g., UDP-3-O-acyl-GlcNAc, m/z 1149.3 > 385.1).
• Quantification: Use external standard curves for semi-quantification. Normalize to cell count or protein content.

4. Data Presentation

Table 1: Comparative Phenotypes: CHIR-090 vs. Genetic lpxC Knockdown

Parameter	CHIR-090 (2 μg/mL)	CRISPRi lpxC KD (50 μM IPTG)	Control (WT)
LpxC mRNA (% of WT)	100% (protein inhibited)	25% ± 5%	100%
LpxC Protein (% of WT)	100% (activity blocked)	30% ± 8%	100%
Growth Inhibition (%)	85% ± 3%	80% ± 6%	0%
Intermediate Accumulation (Fold Change)	12.5x ± 2.1x	10.8x ± 1.9x	1x

Table 2: Conditional Lethality Rescue Assay Results (6h Post-Treatment)

Condition	Final OD600	Viability (CFU/mL)	Intermediate Level
Permissive (+Arabinose)	1.20 ± 0.10	5.2 x 10^8	Baseline
Repressed (+Glucose)	0.15 ± 0.05	2.1 x 10^5	15.3x
Repressed + CHIR-090	0.08 ± 0.03	8.0 x 10^4	18.7x
Repressed + Vector	0.14 ± 0.04	1.9 x 10^5	16.0x
Repressed + LpxC OE	0.95 ± 0.12	3.8 x 10^8	2.1x

5. Diagrams

Validation Workflow for LpxC Targeting

LpxC Inhibition Leads to Toxic Buildup

Application Notes

This document details the comparative safety profile of CHIR-090, a potent, selective inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), against traditional lipopolysaccharide (LPS)-targeting agents such as polymyxins (e.g., colistin) and monoclonal antibodies (e.g., eritoran). The context is the broader thesis research on mitigating the toxicity associated with the accumulation of lipid A pathway intermediates during LpxC inhibition.

• Mechanistic Basis for Differential Toxicity: Traditional LPS-targeting agents like polymyxins bind to and disrupt the outer membrane of Gram-negative bacteria, leading to nephrotoxicity and neurotoxicity due to off-target interactions with eukaryotic membranes. In contrast, CHIR-090 operates upstream, inhibiting the LpxC enzyme in the lipid A biosynthetic pathway. The primary safety concern for LpxC inhibitors is not direct host cell membrane damage but potential intermediate buildup (e.g., UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine). Recent research indicates that CHIR-090's superior pharmacokinetic/pharmacodynamic (PK/PD) profile and chemical scaffold minimize this accumulation, reducing associated cytotoxicity in mammalian cell lines.

• Quantitative Safety Comparison: Data compiled from recent in vitro and preclinical studies highlight the improved therapeutic index of optimized LpxC inhibitors like CHIR-090 derivatives.

Table 1: Comparative Safety and Efficacy Profiles

Parameter	Traditional LPS Agents (Polymyxin B)	LpxC Inhibitor (CHIR-090)	Notes
Primary Target	Lipid A on outer membrane	LpxC enzyme (intracellular)
Key Toxicity	Nephrotoxicity (up to 60% incidence)	Intracellular intermediate accumulation	CHIR-090 structure reduces buildup.
HC50 (μg/mL) [HeLa Cells]	~10-20	>64	Higher HC50 indicates lower cytotoxicity.
MIC90 (μg/mL) [E. coli]	0.5 - 2	0.25 - 1	Comparable potent antibacterial activity.
Therapeutic Index (HC50/MIC90)	~5-40	>64	Significantly wider window for CHIR-090.
Mechanism of Toxicity	Membrane disruption, oxidative stress	Inhibition of cell proliferation (dose-dependent)	CHIR-090 toxicity is reversible upon washout.

HC50: Concentration causing 50% host cell cytotoxicity; MIC90: Minimum Inhibitory Concentration for 90% of isolates.

Protocols

Protocol 1: Assessing Host Cell Cytotoxicity and Lipid Intermediate Accumulation

Objective: To evaluate the safety profile of CHIR-090 by measuring its cytotoxicity and correlating it with intracellular UDP-3-O-acyl-GlcNAc intermediate levels in mammalian cell lines.

Materials: See The Scientist's Toolkit below.

Procedure:

• Cell Seeding: Seed HEK-293 or HeLa cells in 96-well plates at 5 x 10³ cells/well in complete DMEM. Incubate at 37°C, 5% CO₂ for 24 h.
• Compound Treatment: Prepare serial dilutions of CHIR-090 and polymyxin B (positive control) in serum-free medium. Replace medium in wells with compound-containing medium (final concentration range: 0.25 to 128 μg/mL). Include vehicle-only controls. Incubate for 48 h.
• Cytotoxicity Assay (MTT): a. Add 10 μL of MTT reagent (5 mg/mL) to each well. Incubate for 4 h. b. Carefully aspirate medium and add 100 μL of DMSO to solubilize formazan crystals. c. Measure absorbance at 570 nm using a plate reader. Calculate cell viability (%) relative to vehicle control.
• Intermediate Extraction & LC-MS/MS Analysis: a. In parallel, seed cells in 6-well plates. Treat with CHIR-090 at IC₅₀ and IC₉₀ doses for 24 h. b. Wash cells with cold PBS, then scrape into 1 mL of methanol:water (4:1, v/v) at -20°C. c. Sonicate on ice and centrifuge at 16,000 x g for 15 min at 4°C. d. Transfer supernatant and evaporate under nitrogen. Reconstitute in 100 μL mobile phase A. e. Analyze using a reverse-phase C18 column coupled to a tandem mass spectrometer (MRM mode for UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc). Quantify against a synthetic standard curve.

Protocol 2: In Vitro Hemolysis Assay for Membrane Disruption

Objective: To compare the direct membrane-lytic activity of CHIR-090 versus polymyxin B.

Procedure:

• Prepare a 4% (v/v) suspension of fresh, washed human red blood cells (hRBCs) in PBS.
• In a 96-well V-bottom plate, add 100 μL of compound dilutions (in PBS) across a wide concentration range (1-500 μg/mL). Add 100 μL of hRBC suspension. Triton X-100 (1%) and PBS serve as positive and negative controls, respectively.
• Incubate at 37°C for 1 h with gentle shaking.
• Centrifuge the plate at 500 x g for 5 min. Transfer 100 μL of supernatant to a flat-bottom plate.
• Measure absorbance at 540 nm. Calculate % hemolysis = [(Sample Abs - PBS Abs) / (Triton Abs - PBS Abs)] * 100.

Visualizations

Mechanism of Action & Toxicity Pathways

CHIR-090 Cytotoxicity & Intermediate Assay Workflow

The Scientist's Toolkit

Table 2: Essential Research Reagents and Materials

Item	Function/Benefit
CHIR-090 (lyophilized)	Selective, potent LpxC inhibitor; reconstitute in DMSO for in vitro studies.
Polymyxin B sulfate	Traditional LPS-targeting agent; used as a comparative toxicity control.
HEK-293 or HeLa Cell Line	Standard mammalian models for assessing host cell cytotoxicity.
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine	Synthetic analytical standard for quantifying pathway intermediate via LC-MS/MS.
MTT Cell Viability Assay Kit	Colorimetric method to measure metabolic activity and calculate IC₅₀/HC₅₀ values.
C18 Reverse-Phase LC Column	For chromatographic separation of hydrophilic intracellular intermediates.
Triple Quadrupole Mass Spectrometer	Enables sensitive, specific detection and quantification via MRM.
Fresh Human Red Blood Cells	Critical for assessing direct membrane-lytic (hemolytic) activity of compounds.

This application note details protocols for evaluating the synergistic potential of the LpxC inhibitor CHIR-090 in combination with standard-of-care (SoC) antibiotics. LpxC is an essential enzyme in the lipid A biosynthesis pathway of Gram-negative bacteria. CHIR-090's inhibition of LpxC disrupts outer membrane integrity, potentially potentiating the activity of other antimicrobials. This research is framed within a broader thesis investigating CHIR-090's role in reducing intermediate toxicity by precisely targeting a conserved bacterial pathway, thereby creating opportunities for enhanced therapeutic efficacy and reduced antibiotic resistance development.

Key Research Reagent Solutions

The following table lists essential materials for performing synergy assays.

Reagent/Material	Function & Rationale
CHIR-090 (lyophilized)	Potent, selective small-molecule inhibitor of the LpxC enzyme. Reconstitute in DMSO to a 10 mM stock.
Cation-adjusted Mueller Hinton Broth (CAMHB)	Standardized growth medium for antimicrobial susceptibility testing, ensuring reproducible cation concentrations.
Standard-of-Care Antibiotics (e.g., Meropenem, Ciprofloxacin, Tobramycin, Colistin)	Comparator drugs representing different classes (β-lactams, fluoroquinolones, aminoglycosides, polymyxins).
DMSO (Cell Culture Grade)	Sterile solvent for compound reconstitution. Final concentration in assays should not exceed 1% (v/v).
96-well Polypropylene Microplates	For preparing compound dilution series; minimizes compound binding.
96-well Polystyrene Round-Bottom Microtiter Plates	For conducting checkerboard broth microdilution assays.
Resazurin Sodium Salt (0.015% w/v)	Cell viability indicator dye (alamarBlue assay); used for endpoint metabolic readout.
Multichannel Pipettes & Reagent Reservoirs	Essential for efficient liquid handling in high-throughput formats.

Experimental Protocols

Checkerboard Broth Microdilution for Synergy

Objective: To determine the Fractional Inhibitory Concentration Index (FICI) for CHIR-090 in combination with a partner antibiotic.

Protocol:

• Prepare Compound Stocks: Dilute CHIR-090 and the SoC antibiotic in CAMHB to 4x the highest desired test concentration (e.g., 4x MIC).
• Set Up Checkerboard: In a 96-well plate, dispense 50 µL of CAMHB to all wells. Create a two-fold dilution series of CHIR-090 along the x-axis (columns 1-12) and of the SoC antibiotic along the y-axis (rows A-H). This yields a matrix with varying combinations of both agents.
• Inoculate Bacteria: Prepare a bacterial suspension of the target strain (e.g., Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 25922) in CAMHB at ~5 x 10⁵ CFU/mL. Add 50 µL of this suspension to all test wells. Include growth (media + inoculum) and sterility (media only) controls.
• Incubate: Cover plate and incubate statically at 35±2°C for 16-20 hours.
• Determine Endpoints: Visually inspect for turbidity. Alternatively, add 10 µL of resazurin solution (0.015%), incubate for 2-4 hours, and measure fluorescence (Ex/Em: 560/590 nm). The MIC for each agent alone and in combination is the lowest concentration preventing growth/metabolic activity.
• Calculate FICI: For each combination well that inhibits growth, calculate:
  • FIC (CHIR-090) = (MIC of CHIR-090 in combination) / (MIC of CHIR-090 alone)
  • FIC (Antibiotic) = (MIC of Antibiotic in combination) / (MIC of Antibiotic alone)
  • FICI = FIC (CHIR-090) + FIC (Antibiotic)
  • Interpret as: Synergy (FICI ≤ 0.5), Additivity (0.5 < FICI ≤ 1), Indifference (1 < FICI ≤ 4), Antagonism (FICI > 4).

Time-Kill Synergy Assay

Objective: To assess the bactericidal kinetics of the combination over 24 hours.

Protocol:

• Prepare Inoculum: Grow bacteria to mid-log phase (OD600 ~0.5) in CAMHB. Dilute to ~1 x 10⁶ CFU/mL in fresh pre-warmed CAMHB.
• Prepare Treatments: In sterile tubes or deep-well plates, prepare 10 mL volumes containing:
  • Growth control (inoculum only)
  • CHIR-090 at 0.25x, 0.5x, and 1x MIC
  • SoC antibiotic at 0.25x, 0.5x, and 1x MIC
  • Combinations (e.g., CHIR-090 0.25x MIC + SoC 0.25x MIC)
• Sample and Plate: Incubate at 35±2°C with shaking. At T = 0, 2, 4, 6, 8, and 24 hours, remove 100 µL aliquots from each tube, perform serial 10-fold dilutions in sterile saline, and plate 50 µL onto nutrient agar plates.
• Enumerate: Count colonies after overnight incubation. Plot Log10 CFU/mL vs. Time.
• Analysis: Synergy is defined as a ≥2-log10 decrease in CFU/mL by the combination compared to the most active single agent at 24 hours.

Data Presentation

Table 1: Example FICI Results for CHIR-090 Combinations vs.P. aeruginosaPAO1

SoC Antibiotic (Class)	MIC Alone (µg/mL)	MIC in Combination (µg/mL)	FICI	Interpretation
	CHIR-090	SoC	CHIR-090	SoC
Meropenem (β-lactam)	1.0	2.0	0.125	0.5	0.375	Synergy
Ciprofloxacin (FQ)	1.0	0.5	0.25	0.125	0.75	Additivity
Tobramycin (AG)	1.0	1.0	0.5	0.25	1.0	Additivity
Colistin (Polymyxin)	1.0	1.0	2.0	1.0	3.0	Indifference

Table 2: Time-Kill Assay Data (Log10 CFU/mL) at 24 Hours

Treatment Condition	E. coli ATCC 25922	P. aeruginosa PAO1
Growth Control	9.5	9.8
CHIR-090 (1x MIC)	7.2	6.9
Meropenem (1x MIC)	4.1	5.5
CHIR-090 + Meropenem (0.5x MIC each)	1.8	2.3
Change vs. Best Single Agent	-2.3 log10	-3.2 log10
Synergy Outcome?	Yes	Yes

Diagrams

Diagram 1: CHIR-090 & Antibiotic Synergy Mechanism

Diagram 2: Checkerboard Synergy Assay Workflow

Conclusion

CHIR-090 stands as a pivotal proof-of-concept molecule, demonstrating that targeted inhibition of LpxC is a viable and potent strategy for combating Gram-negative infections while strategically mitigating the toxicity associated with pathway intermediate accumulation. The exploration from foundational mechanism through methodological application, troubleshooting, and comparative validation underscores its role as a template for next-generation antibiotic design. Future directions must focus on translating these insights into clinical candidates with optimized pharmacokinetics and resistance profiles, potentially revitalizing the pipeline against multidrug-resistant pathogens. This work solidifies the LpxC target's legitimacy and highlights the continuous need for innovative biochemical strategies in antimicrobial discovery.